Applications of SU3327 in the manufacture of drugs that reduce the cytotoxicity and nephrotoxicity of polymyxins.

Abstract

The use of SU3327, an N-terminal kinase inhibitor of C-JUN, in the manufacture of a drug for reducing the cytotoxicity and nephrotoxicity of polymyxin, wherein the polymyxin is preferably polymyxin E, and the combined use of SU3327 and polymyxin E can significantly inhibit the cytotoxicity and nephrotoxicity of polymyxin E.

Description

Technical Field

[0001] The present invention relates to the field of medicine, specifically to the field of antibacterial agents, and particularly to a method for reducing the cytotoxicity and nephrotoxicity of polymyxin using SU3327.

Background Art

[0002] SU3327 (also called halicin, CAS No.: 40045-50-9) is a kind of C-JUN N-terminal kinase inhibitor. SU3327 is a potent and selective JNK inhibitor with substrate competitiveness, and its IC 50 is 0.7 μM. SU3327 also inhibits the protein interaction between JNK and JIP at an IC 50 value of 239 nM. SU3327 has relatively low activity against p38α and Akt kinases.

[0003] According to the paper "A Deep Learning Approach to Antibiotic Discovery" on pioneer machine learning methods developed by a research team led by synthetic biologist James Collins of the Massachusetts Institute of Technology, it was found that an experiment successfully discovered a new antibiotic, halicin (this research result was published in "Cell" on February 20,

[0004] Polymyxins are antimicrobial peptides discovered in the culture medium of Bacillus polymyxa, and there are five types: A, B, C, D, and E. Their antimicrobial spectra are similar and broad, and they act particularly strongly against Gram-negative bacteria such as Escherichia coli, Neisseria pneumoniae, and Pseudomonas aeruginosa. Polymyxin E (also known as colistin), CAS number: 1066-17-7, English name: Colistin, trade names: Kangujis, Colisticina, and Colistin. Clinical applications are mainly in the form of sulfates or mesylates, i.e., polymyxin E sulfate and polymyxin E mesylate.

[0005] The side effects of polymyxin are mainly expressed in the following ways: (1) Nephrotoxicity, which is a relatively common and relatively serious side effect, can cause a decline in the patient's renal function, and may result in proteinuria, urinary casts, azotemia, and elevated creatinine levels, which are mainly due to impairment of renal function. (2) Certain neurotoxicity, which can cause dizziness, ataxia, drowsiness, and peripheral paresthesia in patients during the period of polymyxin use, and these are all neurological side effects of polymyxin. Nephrotoxicity and neurotoxicity are the most common toxic side effects of the clinical application of polymyxin E, with a high incidence of clinical nephrotoxicity at 60% and neurotoxicity at approximately 7%. Mild peripheral neurotoxicity symptoms are more pronounced and can reach an incidence of 100%. In addition, polymyxin also has clinical application problems such as a "narrow" therapeutic range and the expansion of drug resistance.

[0006] To enhance the therapeutic effects of polymyxins and reduce their cytotoxicity, there is a broad demand in this field for drugs that reduce the cytotoxicity and nephrotoxicity of polymyxins. To date, there have been no research reports on the application of SU3327 in reducing the cytotoxicity and nephrotoxicity of polymyxins. [Overview of the project] [Problems that the invention aims to solve]

[0007] This invention provides a method to reduce the cytotoxicity and nephrotoxicity of polymyxin in order to solve the technical problems of cytotoxicity that occur during treatment with polymyxin. Specifically, it employs a combination of the C-JUN N-terminal kinase inhibitor SU3327 and polymyxin, preferably polymyxin E. The combination of the two not only achieves a synergistic antibacterial effect, but also shows a certain degree of increase in cell viability after combined treatment with SU3327 and polymyxin compared to polymyxin alone. At a certain preferred concentration of SU3327, the combination of the two increased cell viability to 68.5%, which is a significant difference compared to the control. It is shown that when SU3327 and polymyxin E are used in combination, the cytotoxic effect of polymyxin E can be significantly inhibited. Furthermore, by establishing a mouse model of renal damage caused by polymyxin E, we observed that polymyxin E treatment significantly reduced mouse renal function, specifically leading to a significant increase in serum urea nitrogen and creatinine levels, along with clear tubular necrosis, epithelial cell shedding, and the appearance of columnar structures. Concurrent treatment with SU3327 resulted in a significant decrease in serum urea nitrogen and creatinine levels and a clear improvement in tubular pathological changes, demonstrating that concurrent treatment with SU3327 and polymyxin E can significantly reduce the nephrotoxicity caused by polymyxin E. [Means for solving the problem]

[0008] According to one aspect of the present invention, the use of the C-JUN N-terminal kinase inhibitor SU3327 in the manufacture of agents that reduce the cytotoxicity and / or nephrotoxicity of polymyxins is provided.

[0009] According to another aspect of the present invention, the use of a composition of polymyxin and the C-JUN N-terminal kinase inhibitor SU3327 in the manufacture of a drug that reduces the cytotoxicity and / or nephrotoxicity of polymyxin is provided.

[0010] Furthermore, the polymyxin is polymyxin E (i.e., colistin) or polymyxin B.

[0011] Furthermore, the C-JUN N-terminal kinase inhibitor SU3327 and polymyxin E can be prepared as a composition for reducing somatic cytotoxicity or nephrotoxicity in humans or animals caused by polymyxin E.

[0012] Furthermore, the mass ratio of SU3327 to polymyxin in the SU3327 and polymyxin composition is (2.5-10):1.

[0013] Furthermore, the dosage form of the SU3327 and polymyxin composition is one of the following: tablets, capsules, sustained-release tablets, controlled-release tablets, oral solutions, syrups, injectable solutions, dropper pills, or lyophilized powder injections.

[0014] Furthermore, when used to reduce cytotoxicity, the final usable concentration of polymyxin E is 2 mM, and the final usable concentration of SU3327 is 5 μM to 0.625 μM, preferably 2.5 μM.

[0015] Furthermore, concomitant treatment with SU3327 significantly reduced polymyxin E-induced nephrotoxicity. The final dose concentrations of SU3327 used were 2.5 mg / kg body weight / day, 5 mg / kg body weight / day, and 10 mg / kg body weight / day, while the dose of polymyxin E used was 20 mg / kg body weight / day. [Effects of the Invention]

[0016] The novel application of the C-JUN N-terminal kinase inhibitor SU3327 according to the present invention, and the method for reducing polymyxin cytotoxicity, have the following excellent technical effects. (1) The combined use of SU3327 and polymyxin not only achieves a synergistic antibacterial effect, but also shows a certain increase in cell viability compared to polymyxin-only treatment. At a preferred final usage concentration of 2.5 μM for SU3327, the combined use of both increased cell viability to 68.5%, which is significantly higher than the control. When SU3327 is used in combination with polymyxin E, it is shown that the cytotoxic effect of polymyxin E can be significantly inhibited. (2) By establishing a mouse model of renal damage caused by polymyxin E, we demonstrated that treatment with polymyxin E significantly reduced mouse renal function, specifically by a significant increase in serum urea nitrogen and creatinine levels, along with clear tubular necrosis, epithelial cell shedding, and the appearance of columnar structures. After co-treatment with SU3327, serum urea nitrogen and creatinine levels decreased significantly, and tubular pathological changes were clearly improved. This demonstrates that co-treatment with SU3327 and polymyxin E can significantly reduce the nephrotoxicity caused by polymyxin E. (3) The present invention provides a concept to solve the problem of the significant toxic side effects of the clinical application of polymyxin, and can solve technical problems such as the low clinical therapeutic index of polymyxin. [Brief explanation of the drawing]

[0017] [Figure 1] This figure shows the results of reducing the toxicity of polymyxin E to HEK293 cells by SU3327 treatment according to the present invention, and includes the results of detecting cell viability after treatment with SU3327, polymyxin E alone, or in combination. [Figure 2] This figure shows the results of detecting mouse serum urea nitrogen in the control group, polymyxin E model group, SU3327 control group, SU3327 high-dose group, SU3327 medium-dose group, and SU3327 low-dose group of the present invention. [Figure 3] This figure shows the results of mouse serum creatinine detection for the control group, polymyxin E model group, SU3327 control group, SU3327 high-dose group, SU3327 medium-dose group, and SU3327 low-dose group of the present invention. [Figure 4]These are the mouse tissue pathological section diagrams of the control group, polymyxin E model group, SU3327 control group, high-dose SU3327 group, medium-dose SU3327 group, and low-dose SU3327 group of the present invention. [Figure 5] These are the semi-quantitative evaluation diagrams of the control group, polymyxin E model group, SU3327 control group, high-dose SU3327 group, medium-dose SU3327 group, and low-dose SU3327 group of the present invention.

Embodiments for Carrying out the Invention

[0018] The drug adopted in the embodiment of the present invention: SU3327 was purchased from MCE Reagent Company, USA, and its purity is ≧99%. Polymyxin E sulfate was purchased from Hebei Shengxue Dacheng Tangshan Pharmaceutical Company, and its titer is ≧23000 U / mg polymyxin E. Weigh a certain amount of polymyxin E sulfate and prepare it into an aqueous solution with a mother liquor concentration of 16 mg / mL. SU3327 is prepared into a mother liquor with a concentration of 40 mg / mL using DMSO. After all the mother liquors are prepared, they are stored in a refrigerator at -20°C.

Example

[0019] Example 1 Protective effect of SU3327 on the cytotoxicity of polymyxin 1.1 Test cell line Human embryonic kidney cells (HEK293) were preserved by the National Veterinary Drug Safety Evaluation of China Agricultural University.

[0020] 1.2 Detection of cell viability The cytotoxicity was measured by the CCK-8 method, and HEK293 cells (5×10 4After inoculating (cells at a density of) [number of cells] per well into a 96-well tissue culture plate and culturing for 24 hours, cells treated with SU3327 alone, polymyxin E alone, or cells treated with the combination of the two were used. The final concentration of SU3327 was 5 μM, 2.5 μM, 1.25 μM, and 0.625 μM, and the final concentration of polymyxin E was 2 mM. The combined treatment group of SU3327 and polymyxin E had final concentrations of polymyxin E (2 mM) + SU3327 (5 μM), polymyxin E (2 mM) + SU3327 (2.5 μM), polymyxin E (2 mM) + SU3327 (1.25 μM), and polymyxin E (2 mM) + SU3327 (0.625 μM). The control group (Control) was 0.1% DMSO. After continuing the culture for another 24 hours, the change in cell viability was detected using a CCK-8 kit.

[0021] 1.3 Test Results The result figure of the reduction of the toxicity of polymyxin E to HEK293 cells by SU3327 treatment is as shown in Figure 1, and it includes the results of measuring the cell viability by treating with SU3327 alone, polymyxin E alone, or in combination. The specific result analysis is as follows.

[0022] Compared with the control group, the cell viability after treating with 5 μM, 2.5 μM, 1.25 μM, and 0.625 μM of SU3327 alone all showed significant changes. Compared with the control group, after treating with 2 mM of polymyxin for 24 hours, the HEK293 cell viability decreased to 55.3%, showing a significant difference. Compared with the group treated with polymyxin alone, after the combined treatment of SU3327 and polymyxin E, the cell viability showed a certain degree of increase. Among them, when SU3327 was combined with 2.5 μM of polymyxin E for treatment, the cell viability increased to 68.5%, showing a significant difference. It was shown that when SU3327 was combined with polymyxin E for treatment, the cytotoxic effect of polymyxin E could be significantly inhibited.

Example

[0023] Example 2 Protective Effect of SU3327 against Nephrotoxic Injury Induced by Polymyxin in Mice 1 Materials 1.1 Laboratory animals We purchased C57BL / 6 mice, male, 8 weeks old, weighing 20-22g, from Victoria Biotechnology Co., Ltd.

[0024] 2. Test Method 2.1 Rearing and Processing of Laboratory Animals The mice were raised at the National Veterinary Drug Safety Evaluation Center of the College of Animal Medicine, China Agricultural University. They were fed a standard pellet diet, given free access to water, and kept in a controlled environment with a temperature of (22±2)°C and humidity of (50±10)%, adaptively reared for one week prior to the experiment.

[0025] The 30 animals were randomly divided into 6 groups of 8 animals each, as follows: Control group: Mice were injected intraperitoneally with the same amount of physiological saline. Polymyxin E model group: Mice were injected intraperitoneally with polymyxin E. The final single injection dose was 10 mg / kg body weight, administered twice daily at 8-hour intervals, resulting in a total dose of 20 mg / kg body weight / day of polymyxin E per mouse. SU3327 control group: Mice were given intraperitoneal injections of SU3327 dissolved in water. The single injection dose was 10 mg / kg body weight, administered once daily for 10 consecutive days, i.e., mice were given 10 mg / kg body weight / day of SU3327. SU3327 high-dose regimen: Mice are injected intraperitoneally with SU3327, with a single injection dose of 10 mg / kg body weight. Simultaneously, 10 mg / kg body weight of polymyxin E is injected. Polymyxin is then injected twice, every 8 hours, meaning that mice are administered 10 mg / kg body weight of SU3327 per day, and simultaneously 20 mg / kg body weight of polymyxin E. SU3327 medium-dose group: SU3327 is injected intraperitoneally into mice, with a single injection dose of 5 mg / kg body weight. Simultaneously, 5 mg / kg body weight of polymyxin E is injected, and polymyxin is injected twice at 8-hour intervals, meaning that mice are administered 5 mg / kg body weight / day of SU3327 and simultaneously 20 mg / kg body weight of polymyxin E. Low-dose SU3327 group: Mice are injected intraperitoneally with SU3327, with a single injection dose of 2.5 mg / kg body weight. Simultaneously, 10 mg / kg body weight of polymyxin E is injected. Polymyxin is then injected twice, every 8 hours, meaning that mice are administered 2.5 mg / kg body weight / day of SU3327 and simultaneously 20 mg / kg body weight of polymyxin E.

[0026] For the method of establishing a mouse model of polymyxin E nephrotoxicity, please refer to the literature previously published by the inventor (Dai C, Tang S, Wang Y, Velkov T, Xiao X. Baicalein acts as a nephroprotectant that ameliorates colistin-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).

[0027] Ten days after continuous injection administration, 24 hours after the last dose, the mice were euthanized by intraperitoneal injection of an excess of pentobarbital sodium (80 mg / kg), and blood and kidney tissue were rapidly collected. The serum and tissue were then processed as follows.

[0028] 2.2 Measurement of Biochemical Indicators The collected mouse blood samples were centrifuged at 3000 rpm for 10 minutes, and the supernatant was aspirated. Changes in serum urea nitrogen and serum creatinine were detected using an automated biochemical detector.

[0029] 2.3 Renal Histopathology Detection and Semi-Quantitative Scoring After washing the excised fresh kidney tissue with physiological saline, a sample is taken and fixed in 10% neutral formalin. After 48 hours, water is rinsed off, followed by dehydration, clearing, paraffin immersion, embedding, sectioning, and staining. The pathological changes in the kidney tissue are observed and photographed under a microscope. HE staining: The paraffin sections are removed, baked in a 60°C oven for 30 minutes, then dewaxed and dehydrated with ethanol, immersed in double steam water, stained with hematoxylin for 10 minutes, rinsed with tap water, differentiated with hydrochloric acid ethanol, rinsed with running water, and cleared with 95% ethanol for 30 seconds, alcoholic eosin stain for 30 seconds, 95% ethanol for 30 seconds, 100% ethanol for 30 seconds, and xylene until the section turns blue. The sections are then sealed with neutral rubber.

[0030] After HE staining, the degree of kidney tissue damage was evaluated using a semi-quantitative scoring system (SQS). The SQS score was determined by referring to previously published literature (Antimicrob Agents Chemother, 2014 Jul, 58(7):4075-85), and the specific criteria are as follows: SQS = Level score × Damage percentage score, where Level 1 (1 point): Mild tubular damage, i.e., tubular dilation, cell nucleus protrusion, appearance of a small amount of tubular columnar structure; Level 2 (4 points): Severe tubular damage, i.e., tubular epithelial cell necrosis, appearance of a large amount of tubular columnar structure; Level 3 (10 points): Acute necrosis or infarction of the renal cortex or medulla. Kidney section injury percentage score: <1% = 0 points, 1% to <5% = 1 point, 5% to <10% = 2 points, 10% to <20% = 3 points, 20% to <30% = 4 points, 30% to <40% = 5 points, ≥40% = 6 points. Finally, the SQS evaluation results are as follows: SQS+0 = no significant change (total score, <1 point), SQS+1 = mild injury (total score, 1 to <15 points), SQS+2 = between mild and moderate injury (total score, 15 to <30 points), SQS+3 = moderate injury (total score, 30 to <45 points), SQS+1 = between mild and moderate injury (total score, 45 to <60 points), SQS+1 = severe injury (total score, ≥60 points).

[0031] 2.4 Statistical analysis All results are presented as mean ± standard difference. One-way ANOVA was performed using GraphPad 9.0 software, where * represents P < 0.05, ** represents P < 0.01, and **** represents P < 0.001.

[0032] 3. Test Results Compared to the control group, daily injection of polymyxin E at 20 mg / kg for 10 consecutive days significantly increased serum BUN and CRE levels in mice, leading to clear pathological damage including shedding, necrosis, and formation of columnar structures of tubular epithelial cells. The mean pathological semi-quantitative score increased to 3. Compared to the polymyxin E model group, combined treatment with SU3327 at 2.5 mg / kg, 5 mg / kg, and 10 mg / kg with colistin clearly increased serum urea nitrogen (Figure 2 shows the results of detecting urea nitrogen in mice for the control group, polymyxin E model group, SU3327 control group, SU3327 high-dose group, SU3327 medium-dose group, and SU3327 low-dose group) and creatinine (control group, polymyxin E model group). Figure 3 shows the results of detecting mouse serum creatinine levels in the control group, polymyxin E model group, SU3327 control group, SU3327 high-dose group, SU3327 medium-dose group, and SU3327 low-dose group. Figure 4 shows the mouse histopathology section diagrams for the control group, polymyxin E model group, SU3327 control group, SU3327 high-dose group, SU3327 medium-dose group, and SU3327 low-dose group of the present invention, and the corresponding pathological damage scores were reduced to 1 (P<0.001), 0.75 (P<0.001), and 0.5 (P<0.001), respectively. (Figure 5 shows the semi-quantitative evaluation diagrams for the control group, polymyxin E model group, SU3327 control group, SU3327 high-dose group, SU3327 medium-dose group, and SU3327 low-dose group of the present invention). We demonstrated that combining SU3327 with polymyxin E significantly inhibits the nephrotoxicity of polymyxin E.

[0033] The above-described implementations of this disclosure are merely illustrative to illustrate the disclosure and do not limit the embodiments of this disclosure. Those skilled in the art can make other different forms of changes or modifications based on the above description. It is neither necessary nor possible to cover all embodiments comprehensively. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure shall be within the scope of protection of the claims of this disclosure.

Claims

1. A composition comprising SU3327, an N-terminal kinase inhibitor of C-JUN, and polymyxin E, used to reduce nephrotoxicity in the body of humans or animals, wherein the mass ratio of SU3327 to polymyxin is (0.25 to 1):

2.

2. The composition according to claim 1, characterized in that the mass ratio of SU3327 to polymyxin in the composition is 1:

2.

3. The composition according to claim 1 or 2, characterized in that the composition is in one dosage form from among tablets, capsules, sustained-release tablets, controlled-release tablets, oral solutions, syrups, injectable solutions, drop-type pills, and lyophilized powder injections.

4. The composition according to claim 1, characterized in that, when used to reduce nephrotoxicity, the final dose concentration of SU3327 is 2.5 to 10 mg / kg body weight / day, and the final dose of polymyxin E is 20 mg / kg body weight / day.

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