Use of sphingosine in the preparation of a medicament for the treatment of a bacterial infectious disease or an antibiotic potentiator

Abstract

The application discloses application of sphingosine or its acceptable salt in preparation of a medicine or preparation for killing and / or inhibiting bacteria in and out of organisms. The application discloses application of sphingosine or its acceptable salt in preparation of a medicine for bacterial infectious diseases or preparation of an antibiotic synergist. The application discloses a medicine composition comprising sphingosine and colistin. The application discloses application of the composition in preparation of a medicine or preparation for killing and / or inhibiting bacteria in and out of organisms or preparation of a medicine for bacterial infectious diseases or an antibiotic synergist. The application finds the possibility of sphingosine as the colistin synergist for resisting multiple drug-resistant pathogenic bacterial infection through in-vivo and in-vitro antibacterial tests. The sphingosine can reduce the minimum inhibitory concentration of colistin to drug-resistant gram-negative bacteria by 30 times, greatly improving the antibacterial activity of colistin.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of sphingosine in the preparation of drugs for treating bacterial infectious diseases or as an antibiotic potentiator. Background Technology

[0002] With the emergence and spread of multidrug-resistant bacteria, bacterial resistance to antibiotics poses a serious threat to global public health. Recent reports indicate a high number of deaths worldwide related to bacterial AMRs, particularly in Africa where healthcare is relatively underdeveloped. Carbapenem-producing Enterobacteriaceae are among the most significant drug-resistant bacteria threatening human and animal health. Colistin, a cationic cyclic peptide antibiotic, is a last resort against carbapenem-producing Enterobacteriaceae. Colistin exerts its antibacterial effect by binding to the lipopolysaccharide of the outer membrane of Gram-negative bacteria, mediating cell membrane rupture, leakage of intracellular contents, and ultimately bacterial lysis. However, the transferable colistin resistance gene (mcr-1), encoding phosphoethanolamine transferase, reduces the negative charge of lipid A and mediates acquired colistin resistance. Subsequently, other mcr variants, including mcr-2-10, were quickly discovered, and their prevalence has been greatly enhanced due to their plasmid-borne nature and horizontal spread. Currently, the positivity rate of mcr-1 and its variants is as high as 45%, which greatly challenges the effectiveness of colistin, leading to treatment failure in carbapenem-producing Enterobacteriaceae infections. Therefore, there is an urgent need to overcome MCR-mediated colistin resistance. Due to high costs, long development cycles, and low profitability, the pipeline for developing new antibiotics has been exhausted since the late 1990s, and the development speed of new antibacterial drugs has lagged far behind the development speed of resistance, ushering in a post-antibiotic era. In contrast, antibiotic adjuvants overcome antibiotic resistance by slowing the development of resistance and improving the efficacy of antibiotics, making them a cost-effective strategy against multidrug-resistant (MDR) bacteria. Clavulanic acid, a β-lactamase inhibitor, is a prime example of success, largely salvaging the efficacy of penicillin and cephalosporin antibiotics. Therefore, developing colistin adjuvants is an important means of combating colistin resistance.

[0003] Sphingosine is an unsaturated C18 aminodiol, primarily produced by the enzymatic hydrolysis of sphingomyelin from foods such as meat, eggs, and dairy. The stratum corneum of the human epidermis also contains abundant ceramides (sphingosine derivatives). When the skin barrier is damaged or infected, acidic sphingomyelinase in keratinocytes is activated, hydrolyzing sphingomyelin and releasing sphingosine in situ. This locally produced sphingosine can reach effective antibacterial concentrations, acting as a rapid-response chemical defense weapon to help eliminate invading pathogens and is an important component of the skin's innate immune system. Previous studies have found that sphingosine has good activity against Gram-positive bacteria; however, research on its synergistic effect with colistin against Gram-negative bacteria remains limited. Summary of the Invention

[0004] Objective of the Invention: To address the problems existing in the prior art, this invention aims to explore the synergistic effect, antibacterial properties, synergistic mechanism, and potential value of combined in vivo application of sphingosine on colistin. Based on this, the invention provides a feasible technical solution to address the increasingly serious problem of drug resistance, effectively enhance the efficacy of colistin, and extend the clinical use period of colistin.

[0005] The technical problem to be solved by the present invention is to provide the use of sphingosine or an acceptable salt thereof in the preparation of medicaments for bacterial infectious diseases or in the preparation of antibiotic potentiators.

[0006] Another technical problem to be solved by the present invention is to provide a therapeutic combination of sphingosine and polymyxin that can synergistically kill or fight bacteria in vivo or in vitro.

[0007] Another technical problem to be solved by the present invention is to provide the use of the composition in the preparation of medicaments for bacterial infectious diseases.

[0008] Technical solution: In order to solve the above-mentioned technical problems, the present invention provides the use of sphingosine or an acceptable salt thereof in the preparation of drugs or preparations for killing and / or inhibiting bacteria in vivo and in vitro.

[0009] The present invention also provides the use of sphingosine or an acceptable salt thereof in the preparation of medicaments for bacterial infectious diseases or in the preparation of antibiotic potentiators.

[0010] The drug, preparation, or antibiotic synergist mentioned herein is a single-component or compound preparation.

[0011] The bacteria are Gram-positive or Gram-negative; preferably, the bacteria are drug-resistant; preferably, the bacteria include one or more of Escherichia coli ATCC 25922, Escherichiacoli K582 (mcr-10), Salmonella SDJ02 (mcr-9), E. coli B2 (mcr-1), E. coli G92 (mcr-1), E. coli BL-21-pET28a-mcr-1, Salmonella 15E343 (mcr-3), and Klebsiella pneumoniae 19-2-1 (mcr-8).

[0012] The antibiotic in question is colistin.

[0013] The present invention also provides a pharmaceutical composition comprising sphingosine and colistin.

[0014] The concentration of sphingosine is 0.5~64 μg / mL.

[0015] The concentration of the colistin is 0.03~4 μg / mL.

[0016] The mass ratio of sphingosine to colistin is 1:1 to 8:1.

[0017] The present invention also provides the use of the described composition in the preparation of pharmaceuticals or formulations for killing and / or inhibiting bacteria in vivo and in vitro, or in the preparation of pharmaceuticals or antibiotic potentiators for bacterial infectious diseases.

[0018] The dosage form of the drug or antibiotic potentiator for bacterial infectious diseases is tablet, capsule, oral liquid, syrup, drop pill, injection, or lyophilized powder for injection.

[0019] Beneficial Effects: Compared with existing technologies, the advantages of this invention are: This invention provides the use of sphingosine as an antibacterial drug, restoring the sensitivity of colistin-resistant bacteria, and is one of the strategies for solving the problem of bacterial resistance. This invention, through in vitro and in vivo antibacterial tests, discovers the potential of sphingosine as a colistin potentiator against multidrug-resistant pathogens. Sphingosine can reduce the minimum inhibitory concentration of colistin against drug-resistant Gram-negative bacteria by up to 30 times, significantly improving the antibacterial activity of colistin. Attached Figure Description

[0020] Figure 1The study aimed to verify the in vitro synergistic activity of sphingosine (DES) and colistin (COL) through combined drug susceptibility testing. Results showed that sphingosine enhanced the antibacterial activity of colistin against both resistant and susceptible bacteria, with FIC values ​​below 0.5. Sphingosine reduced the MIC of colistin against resistant bacteria by more than 30-fold.

[0021] Figure 2 For the combined drug susceptibility test of E. coli B2, the synergistic effect was significant, with FICI < 0.1. Unless otherwise specified, this bacterium was used as the research object in subsequent experiments.

[0022] Figure 3 Bactericidal curve assays revealed that sphingosine significantly enhanced the bactericidal activity of colistin against drug-resistant bacteria. For both logarithmic-phase and plateau-phase bacteria, the combination of sphingosine and colistin reduced the bacterial load of multidrug-resistant *Escherichia coli* B2 (mcr-1) to the limit of detection (10⁻⁶). 2 Colistin (CFU / mL) can achieve bactericidal activity when its concentration is below a certain level, while colistin alone cannot achieve bactericidal activity.

[0023] Figure 4 Survival tests on the giant wax moth showed that sphingosine significantly improved the in vivo antibacterial effect of colistin, increasing the survival rate of the giant wax moth from 0% to 80%.

[0024] Figure 5 In mouse infection survival tests, sphingosine significantly improved the in vivo antibacterial effect of colistin, increasing the mouse survival rate from 30% to 90%.

[0025] Figure 6 In a mouse peritonitis infection model, the combined use of sphingosine and colistin significantly reduced the bacterial load in vivo by about 100 times, demonstrating a good synergistic effect in vivo.

[0026] Figure 7 The hemolysis assay showed that sphingosine had a hemolytic activity of less than 20% at a concentration of 512 μg / ml, indicating good biocompatibility. Detailed Implementation

[0027] The present invention will be further illustrated below with specific embodiments. It should be noted that those skilled in the art can make various modifications and improvements without departing from the principles of the present invention, and these should also be considered within the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores. All quantitative experiments in the following embodiments were performed in triplicate, and the results were averaged.

[0028] Relevant strains of the present invention are derived from the following articles: 1. Liu Y, Jia Y, Yang K, Tong Z, ShiJ, Li R, Xiao X, Ren W, Hardeland R, Reiter RJ, Wang Z. Melatonin overcomesMCR-mediated colistin resistance in Gram-negative pathogens. Theranostics.2020 Aug 29;10(23); 2. Huang Y, Wang Z, Liu Z, Huan Q, Liu Y, Li R, Wang M,Xiao

[0029] Example 1: Synergistic antibacterial activity of sphingosine and colistin

[0030] The checkerboard dilution method was used to determine the synergistic antibacterial activity of sphingosine and colistin in combination against susceptible Escherichia coli ATCC 25922, Escherichia coli K582 (mcr-10), Salmonella SDJ02 (mcr-9), and drug-resistant Escherichia coli E. coli B2 (mcr-1), E. coli G92 (mcr-1), E. coli BL21 (pET28a-mcr-1), Salmonella 15E343 (mcr-3), and Klebsiella pneumoniae 19-2-1 (mcr-8).

[0031] The specific steps of the chessboard dilution method are as follows:

[0032] (1) Dilute the bacterial suspension of the test strain with CAMHB broth medium (Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., HB6231-1) to make the bacterial suspension concentration 1×10⁻⁶. 6 CFU / mL.

[0033] (2) Colistin was dissolved in distilled water and diluted with CAMHB broth medium to obtain a colistin solution with a concentration of 32 μg / mL.

[0034] (3) Sphingosine was dissolved in DMSO and diluted with CAMHB broth medium to obtain a sphingosine solution with a concentration of 256 μg / mL.

[0035] (4) Take a 96-well plate and add 100 μL of CAMHB broth medium to each well. Add 100 μL of the colistin solution prepared in step (2) to each well in the first row. Serial dilute from the first row to the seventh row. The concentration gradient of colistin after dilution is 8, 4, 2, 1, 0.5, 0.25, 0.12, 0 μg / mL. Add 100 μL of the sphingosine solution prepared in step (3) to each well in the first column. Serial dilute to the seventh column. The concentration gradient after dilution is 128, 64, 32, 16, 8, 4, 2, 0 μg / mL. Then add 100 μL of the bacterial suspension prepared in step (1) to each well. Incubate at 37℃ for 16 h-20 h and observe the lowest concentration combination of sphingosine and colistin that inhibits bacterial growth.

[0036] The method for calculating fractionated inhibitory concentration (FICI) is as follows:

[0037] FIC = MIC (drug A in combination) / MIC (drug A alone) + MIC (drug B in combination) / MIC (drug B alone);

[0038] Experimental results are as follows Figure 1 As shown, sphingosine can enhance the bactericidal activity of colistin against colistin-resistant bacteria mediated by chromosomal mutations, those mediated by the mcr gene, and susceptible bacteria, with FIC values ​​all below 0.5. Figure 1 This indicates that sphingosine is a broad-spectrum synergist of colistin. It is particularly effective against E. coli B2, with a FICI value less than 0.1 (…). Figure 2 ).

[0039] Example 2: Time-based bactericidal curve of sphingosine combined with colistin

[0040] Drug-resistant E. coli B2 (mcr-1) was cultured in CAMHB broth at 37°C to the exponential and stationary phases, and the bacterial culture was diluted with CAMHB broth to a final concentration of 10⁻⁶. 7The concentration of CFU / mL was determined. Bacteria were then treated alone or in combination with sphingosine (final concentration 16 μg / mL) and colistin (final concentration 2 μg / mL). 50 μL of bacterial culture was taken at 0, 4, 8, 12, and 24 h, serially diluted 10-fold, and dropped onto LB agar plates. After incubation at 37°C for 24 h, colony forming units (CFU / mL) were calculated. All experiments were performed at least three times.

[0041] The results are as follows Figure 3 , Figure 3 The results showed that the combined use of sphingosine (16 μg / mL) and colistin (2 μg / mL) could kill bacteria, reducing the bacterial load by more than 4 log values. Figure 3 These results further demonstrate that sphingosine can indeed significantly enhance the bactericidal activity of colistin against drug-resistant bacteria.

[0042] Example 3: In vivo antibacterial activity of sphingosine and colistin - survival rate of large wax moth larvae

[0043] Single colonies of drug-resistant *E. coli* B2 (mcr-1) were picked and cultured overnight in 1 mL of blank MHB at 37°C and 200 rpm. The next day, the bacterial suspension was diluted 1:1000 in blank MHB for expansion culture, and resuspended in PBS after 4 hours. Forty large wax moth larvae (300 mg, purchased from Tianjin Huiyude Co., Ltd.) were prepared and revived in a 37°C incubator. After 4 hours, they were randomly divided into 4 groups and placed in 9 cm agar plates. *E. coli* suspension was administered via a microsyringe through the right hind leg. One hour after infection, treatment was administered via injection of PBS, colistin alone (1 mg / kg), sphingosine alone (2 mg / kg), and a combination of colistin and sphingosine (1 mg / kg + 2 mg / kg) into the left hind leg. All large wax moths were placed in a 37°C incubator and observed for 5 days. The survival rate was calculated based on the number of surviving larvae in each group. Results are as follows: Figure 4 As shown, sphingosine significantly enhanced the in vivo antibacterial effect of colistin, increasing the survival rate of the giant wax moth from 0% to 80%.

[0044] Example 4: In vivo antibacterial activity of sphingosine and colistin - mouse intraperitoneal infection model

[0045] Single colonies of drug-resistant E. coli B2 (mcr-1) were picked and placed in 1 mL of blank MHB and incubated overnight at 37°C and 200 rpm. The next day, the bacterial culture was diluted 1:100 in blank MHB for expansion culture, and after 4 hours, it was resuspended in PBS to OD. 600=0.6 for later use. Thirty-two female ICR mice (6 weeks old, 20 g, selected from the Comparative Medicine Center of Yangzhou University) were randomly divided into four groups. All mice were intraperitoneally injected with 0.1 mL of *E. coli* suspension using a syringe. One hour after infection, they were treated with 0.1 mL of PBS (0.01 M, pH 7.4), colistin alone (0.9 mg / kg), sphingosine alone (1.7 mg / kg), or a combination of both (colistin (0.9 mg / kg) + sphingosine (1.7 mg / kg)) intraperitoneally. Survival rate was observed within 60 hours. Simultaneously, viscera were collected, ground, and subjected to bacterial colony counting. The mouse infection survival rate experiment showed that sphingosine significantly enhanced the in vivo antibacterial effect of colistin, increasing the mouse survival rate from 30% to 90%. Figure 5 In a mouse peritonitis infection model, the combined use of sphingosine and colistin significantly reduced the bacterial load in vivo by approximately 100-fold, demonstrating a good synergistic effect in vivo. Figure 6 ).

[0046] Example 5: Sphingosine toxicity assay - hemolytic test

[0047] Spread 100 mL / well of PBS (0.01 M, pH 7.4) across columns 1 to 6 of a 96-well U-shaped plate. Add 100 μL / well of double-distilled water to column 7 as a positive control, and add 100 μL / well of PBS to column 8 as a negative control. Add 100 μL / well of sphingosine (final concentration 1024 μg / mL) to column 1, serially diluting it to column 6. The resulting concentration gradients are 1024, 512, 256, 128, 64, and 32 μg / mL. Discard any excess liquid. Collect anticoagulated sheep blood (Haibo Biotechnology, HB0124), centrifuge at 3000×g for 10 minutes, discard the supernatant, and resuspend in an equal volume of PBS. Repeat this process twice to obtain a sheep red blood cell suspension. Add 1.6 mL of sheep red blood cells to 18.4 mL of PBS to prepare an 8% red blood cell suspension. 100 μL / well of 8% erythrocyte suspension was spread across the first to eighth columns of a 96-well U-shaped plate. The plate was incubated at 37°C for 1 hour. 120 μL of the reaction solution was transferred to a new 0.5 mL centrifuge tube from each well, centrifuged at 3,000g for 5 min, and 100 μL was added to the U-shaped plate. The absorbance at 576 nm was measured using a microplate reader. The hemolysis rate was calculated using the formula: Hemolysis (%) = [(OD576)] sample -OD576 blank ) / (OD576 H2O -OD576 blank ) × 100%. The result is as follows: Figure 7As shown, the hemolytic assay revealed that celandine had a hemolytic activity of less than 15% at a concentration of 512 μg / ml, indicating good biocompatibility.

Claims

1. The use of sphingosine or an acceptable salt thereof in the preparation of drugs or formulations for killing and / or inhibiting bacteria in vivo and in vitro.

2. Use of sphingosine or an acceptable salt thereof in the preparation of medicaments for bacterial infectious diseases or in the preparation of antibiotic potentiators.

3. The application according to claim 1 or 2, characterized in that, The drug, preparation, or antibiotic potentiator mentioned is a single-component or compound preparation.

4. The application according to claim 1 or 2, characterized in that, The bacteria are Gram-positive or Gram-negative bacteria; preferably, the bacteria are drug-resistant bacteria; preferably, the bacteria include one or more of the following: *Escherichia coli* ATCC 25922, *Escherichia coli* K582 (mcr-10), *Salmonella* SDJ02 (mcr-9), *E. coli* B2 (mcr-1), *E. coli* G92 (mcr-1), *E. coli* BL-21-pET28a-mcr-1, *Salmonella* 15E343 (mcr-3), and *Klebsiella pneumoniae* 19-2-1 (mcr-8).

5. The application according to any one of claims 1 to 4, characterized in that, The antibiotic in question is colistin.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes sphingosine and colistin, and preferably, the concentration of sphingosine is 0.5~64 μg / mL.

7. The pharmaceutical composition according to claim 6, characterized in that, The concentration of the colistin is 0.03~4 μg / mL.

8. The pharmaceutical composition according to claim 6, characterized in that, The mass ratio of sphingosine to colistin is 1:1 to 8:

1.

9. The use of the composition according to any one of claims 6 to 8 in the preparation of a medicament or preparation for killing and / or inhibiting bacteria in vivo and in vitro, or in the preparation of a medicament or antibiotic potentiator for bacterial infectious diseases.

10. The application according to claim 9, characterized in that, The dosage form of the drug or antibiotic potentiator for bacterial infectious diseases is tablet, capsule, oral liquid, syrup, drop pill, injection, or lyophilized powder for injection.

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