Application of Obtoclax mesylate and / or Nigericin in preparation of antibiotic sensitizer

Abstract

The invention relates to the technical field of biological medicine. The invention provides an application of Obtoclax mesylate and / or Nigericin in preparation of an antibiotic sensitizer, wherein the antibiotic comprises colistin. The invention also provides an application of Obtoclax mesylate and / or Nigericin in preparation of the antibiotic sensitizer. The invention further provides an application of the medicine composition of the colistin and the Obtoclax mesylate or the Nigericin in preparation of the medicine for resisting the colistin drug resistance gram-negative bacteria, and the medicine composition of the colistin and the Obtoclax mesylate or the Nigericin can be used for preparing the medicine. According to the technical scheme, the colistin and Obtoclax mesylate or Niguricin are combined for use, so that a synergistic effect is achieved, the MIC value of the colistin for resisting colistin-resistant gram-negative bacteria can be remarkably reduced, and the growth of the gram-negative bacteria is inhibited.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of obatoclax mesylate and / or nigericin in the preparation of antibiotic sensitizers. Background Technology

[0002] Colistin is a last-line treatment for multidrug-resistant Klebsiella pneumoniae. Clinically, it is often used in combination with other antibiotics to treat severe carbapenem-resistant Klebsiella pneumoniae. K. pneumoniae Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are increasingly prevalent. However, with the increased use of colistin, colistin-resistant CRKP strains have emerged, with a resistance rate of approximately 4%. More seriously, most colistin-resistant CRKP strains are highly virulent ST11 strains, these "superbugs" resistant to almost all antibiotics except tigecycline and ceftazidime / avibactam. Extensive clinical data show that tigecycline is ineffective in treating CRKP infections, and both colistin and tigecycline pressure can rapidly induce carbapenem-resistant highly virulent Klebsiella pneumoniae (CR-hvKP) to develop colistin resistance in a short period. Currently, the World Health Organization has listed CRKP as a top priority for the research, discovery, and development of novel antimicrobial drugs. Therefore, overcoming the challenge of colistin resistance in CRKP has become an urgent need and core task in current clinical treatment.

[0003] With the problem of bacterial resistance becoming increasingly serious, coupled with the long development cycle, high difficulty, and sharp decline in the number of approved new antibiotics, exploring new uses for existing drugs and developing highly effective antibiotic sensitizers have become key strategies for dealing with drug-resistant bacterial infections and overcoming clinical treatment dilemmas. Summary of the Invention

[0004] The purpose of this invention is to provide an application of obatoclax mesylate and / or nigericin in the preparation of antibiotic sensitizers. The combined use of obatoclax mesylate or nigericin with colistin can effectively enhance the effect of colistin, thereby achieving a better effect of inhibiting the growth of colistin-resistant Gram-negative bacteria.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the use of obatoclax mesylate and / or nigericin in the preparation of antibiotic sensitizers.

[0006] Preferably, the antibiotic includes colistin.

[0007] The present invention also provides the use of a pharmaceutical composition of colistin with the aforementioned Obatoclax mesylate or Nigericin in the preparation of an anticolistin-resistant Gram-negative bacterial drug.

[0008] Preferably, the Gram-negative bacteria include Escherichia coli and Klebsiella pneumoniae.

[0009] Preferably, the concentration of colistin in the pharmaceutical composition is 4-64 mg / L.

[0010] Preferably, the concentration of Obatoclax mesylate in the pharmaceutical composition is 0.032-512 mg / L.

[0011] Preferably, the concentration of Nigericin in the pharmaceutical composition is 0.032-512 mg / L.

[0012] By adopting the above technical solution, the present invention has the following beneficial effects: This invention uses a checkerboard method to screen various existing ion carriers, identifying a synergistic effect between obatoclax mesylate or nigericin and colistin, thus exploring its novel application as an antibiotic sensitizer in the treatment of drug-resistant bacteria. The invention also demonstrates through bactericidal curves and drug susceptibility testing that the combined use of obatoclax mesylate or nigericin and colistin can inhibit the growth of colistin-resistant Gram-negative bacteria, significantly reducing the MIC value of colistin against these bacteria, including *Escherichia coli* and *Klebsiella pneumoniae*. In other words, the obatoclax mesylate or nigericin described in this invention can reverse colistin resistance.

[0013] The Obatoclax mesylate and Nigericin described in the technical solution of this invention are both anti-tumor drugs, which have good anti-cancer activity against a variety of cancers, especially against tumor stem cells that are highly resistant to chemotherapy and cause tumor recurrence. Attached Figure Description

[0014] Figure 1 Figure 1 shows the in vitro bactericidal curves for different treatment groups. Figure 1 In the diagram, (a) represents the bactericidal effect of the Nigericin and Nigericin combined treatment group, and (b) represents the bactericidal effect of the Obatoclax mesylate and Obatoclax mesylate combined treatment group. Detailed Implementation

[0015] This invention provides the use of Obatoclax mesylate (catalog number 010012, Beijing Puxitang Biotechnology Co., Ltd.) and / or Nigericin (catalog number N60011, Beijing Puxitang Biotechnology Co., Ltd.) in the preparation of antibiotic sensitizers.

[0016] In this invention, the antibiotic is preferably colistin.

[0017] The present invention also provides the use of a pharmaceutical composition of colistin with the aforementioned Obatoclax mesylate or Nigericin in the preparation of an anticolistin-resistant Gram-negative bacterial drug.

[0018] In this invention, the Gram-negative bacteria include Escherichia coli and Klebsiella pneumoniae.

[0019] In this invention, the concentration of colistin in the pharmaceutical composition is preferably 4-64 mg / L.

[0020] In this invention, the concentration of Obatoclax mesylate in the pharmaceutical composition is preferably 0.032-512 mg / L, and more preferably 4 mg / L.

[0021] In this invention, the concentration of Nigericin in the pharmaceutical composition is preferably 0.032-512 mg / L, and more preferably 4 mg / L.

[0022] 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.

[0023] Example 1: Screening of ion carriers with in vitro synergistic effects with colistin

[0024] (a) Chessboard Method

[0025] Fresh single colonies of CRKP C2582 strain (Zhang Y, Wang X, Wang S, Sun S, LiH, Chen H, Wang Q and Wang H (2021) Emergence of Colistin Resistance in Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae Under the Pressure of Tigecycline. Front. Microbiol. 12: 756580. doi: 10.3389 / fmicb.2021.7565) were picked and inoculated into 3 mL of physiological saline. The bacterial suspension concentration was adjusted to 0.5 McFarland turbidity, and then diluted 1:100 with MH broth. Based on the previously determined minimum inhibitory concentration (MIC) of the antimicrobial agents, a concentration gradient for combined antimicrobial susceptibility testing was set. Using the microbroth dilution method, 50 μL each of antimicrobial solutions A and B at different concentration gradients, and 100 μL of the diluted bacterial suspension were added to 96-well plates. Drug A is colistin (concentration range 0.5~64 mg / L), serially diluted twofold along the X-axis of a 96-well plate; Drug B is the ion carrier to be tested (concentration range 0.032~512 mg / L), serially diluted twofold along the Y-axis. The compounds to be tested included: Amphotericin B (catalog number SL3980, Beijing Cooler Technology Co., Ltd.), EGCG (catalog number CE5042, Beijing Cooler Technology Co., Ltd.), Hydroxy-chloroquine (catalog number H0915, Sigma Aldrich), Ivermycin (catalog number I10207, MedChemExpress LLC), Natamycin (catalog number HY-B0133, MedChemExpress LLC), Nystatin (catalog number N6261, Sigma Aldrich), Monensin sodium salt (catalog number A735800, Kemeike (Shanghai) Pharmaceutical Technology Co., Ltd.), Curcumin (catalog number 08511, Sigma Aldrich), Nigericin (catalog number N60011, Beijing Puxitang Biotechnology Co., Ltd.), Salinomycin (catalog number HY-15597, MedChemExpress LLC), and Obatoclax. mesylate (product number 010012, Beijing Puxitang Biotechnology Co., Ltd.) and Gramicidin (product number HY-P0163, MedChemExpress LLC).After sample addition, incubate at 35 - 37 °C for 16 - 18 h, then read and record the MIC value.

[0026] Calculate the fractional inhibitory concentration index (FICI) according to the following formula: FICI = (MIC of drug combination of A and B / MIC of drug A alone) + (MIC of drug combination of A and B / MIC of drug B alone).

[0027] When FICI ≤ 0.5, it indicates synergistic effect; when 1 < FICI ≤ 4, it indicates no relevant effect; when FICI > 4, it indicates antagonistic effect.

[0028] Table 1 Screening of ionophores with synergistic effect with colistin

[0029] The results showed that among the 12 ionophores screened, the ionophores Obatoclax and Nigericin could significantly reverse the resistance of CRKP to colistin, reducing the MIC of colistin against this strain to ≤ 1 mg / L (Table 1).

[0030] (2) Bactericidal curve

[0031] Inoculate the above-mentioned CRKP strain onto a blood agar plate and incubate at 35 °C for 16 - 18 h; pick a fresh single colony into 5 mL of LB broth and culture it with shaking at 37 °C and 200 rpm until the logarithmic growth phase (about 4 h). Take the bacterial suspension and co-culture it with the drug groups at 37 °C and 200 rpm. Set up a control group (without antibacterial drug, LB broth), single drug A group (colistin 2 mg / L, denoted as Colistin), single drug B group (Obatoclax mesylate 4 mg / L or Nigericin 4 mg / L), and drug A + B combination group (colistin 2 mg / L + Obatoclax mesylate 4 mg / L; or colistin 2 mg / L + Nigericin 4 mg / L). Add 5 μL of the bacterial suspension (final concentration is 10 5 CFU / mL) to each group, mix well by shaking, then place it in a shaker at 37 °C and 220 rpm. Take 50 μL of the bacterial liquid at 0, 2, 4, 8, and 24 h respectively, dilute it, and then take 50 μL and spread it evenly on an MH agar plate. After incubating at 35 °C for 16 - 18 h, perform colony counting. Set up two biological replicates for each group.

[0032] The results are as Figure 1 shown. After Nigericin and Obatoclax mesylate are combined with colistin respectively, both can significantly inhibit the growth of colistin-resistant CRKP.

[0033] Example 2

[0034] Based on the ion carriers Obatoclax mesylate and Nigericin obtained from the above screening, this study further expands their antibacterial sensitization effects. In addition to CRKP, their resistance reversal effect on colistin-resistant Escherichia coli was investigated.

[0035] Common and clinically significant Gram-negative bacteria, such as Klebsiella pneumoniae and Escherichia coli, were selected as research strains. All strains were identified by MALDI-TOF MS (Bruker Daltonics, Billerica, MA, USA). Following the CLSI guidelines, the minimum inhibitory concentration (MIC) of colistin alone and in combination with ionotropic agents was determined using the microbroth dilution method. The specific procedure was as follows: a fresh single colony was picked and placed in 3 mL of physiological saline, and the bacterial suspension concentration was adjusted to 0.5 McFarland turbidity. The bacterial suspension was then diluted 1:100 with MH broth. The MIC of the test bacteria in the following MH broth systems were determined: ① Colistin only (concentration range 0.5 mg / L–64 mg / L); ② Colistin (0.5 mg / L–64 mg / L) combined with 4 mg / L Obatoclax mesylate or 4 mg / L Nigericin, respectively; ③ Colistin only (4 mg / L Obatoclax mesylate) or Nigericin only.

[0036] Table 2. Effects of Nigericin or Obatoclax mesylate combined with colistin on the colistin MIC of colistin-resistant Gram-negative bacteria.

[0037] Note: Eco Indicates Escherichia coli; Kpn This indicates Klebsiella pneumoniae.

[0038] The strains GZ1 and GZ2 mentioned in Table 2 have been described in the literature "Decreased Fitness and Virulence in ST10". Escherichia coli Harboring bla NDM-5 and mcr-1 against a ST4981 Strain with bla NDM-5”Statement (Zhang Y, Liao K, Gao H, Wang Q, Wang X, Li H, Wang R, Wang H.Decreased fitness and virulence in ST1 Escherichia coli Harboring bla NDM-5 and mcr-1 against a ST4981 Strain with bla NDM-5 . . . . Front Cell Infect Microbiol. 2017Jun 8;7:242.)NonflowerC2582“Emergence of Colistin Resistance inCarbapenem-Resistant Hypervirulent Klebsiella pneumoniae Zhang Y, Wang X, Wang S, Sun S, Li H, Chen H, Wang Q,Wang H. Emergence of Colistin Resistance in Carbapenem-ResistantHypervirulent Klebsiella pneumoniae Under the Pressure of Tigecycline. FrontMicrobiol. 2021 Dec 1;12:756580.)(Elucidating adaptivecompensatory tigecycline resistance mechanisms of RamA, RarA and SoxS in the HK1-based system). Klebsiella pneumoniae ”(Kuai J, Zhao Y, Wang R, Zhang Y, Li H, Chen H,Wang X, Wang H. Elucidating adaptive compensatory tigecycline resistancemechanisms of RamA, RarA and SoxS in Klebsiella pneumoniae. Int J AntimicrobAgents Oct;66(4):107551.)

[0039] The results showed that Obatoclax mesylate and Nigericin could also reduce the colistin MIC in colistin-resistant Escherichia coli (Table 2).

[0040] In summary, the technical solution described in this invention has a synergistic effect when colistin is used in combination with obatoclax mesylate or nigericin, which can inhibit the growth of colistin-resistant CRKP and Escherichia coli, and significantly reduce the MIC value of colistin against colistin-resistant Gram-negative bacteria.

[0041] 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 obatoclax mesylate and / or nigericin in the preparation of antibiotic sensitizers, characterized in that, The antibiotics include colistin.

2. The use of a pharmaceutical composition of colistin and the Obatoclax mesylate or Nigericin as described in claim 1 in the preparation of an anticolistin-resistant Gram-negative bacterium drug.

3. The application according to claim 2, characterized in that, The Gram-negative bacteria include colistin-resistant Escherichia coli and Klebsiella pneumoniae.

4. The application according to claim 2, characterized in that, The concentration of colistin in the pharmaceutical composition is 4-64 mg / L.

5. The application according to claim 2, characterized in that, The concentration of Obatoclax mesylate in the pharmaceutical composition is 0.032-512 mg / L; The concentration of Nigericin in the pharmaceutical composition is 0.032-512 mg / L.

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