Use of paclitaxel in enhancing the antibacterial effect of tigecycline

Abstract

The present application relates to the new use of paclitaxel in the synergistic effect of the antibiotic action of tigecycline, and the use of paclitaxel in the preparation of a composition or a compound preparation for the synergistic effect of the antibiotic action of tigecycline, and the ratio of paclitaxel to tigecycline is between 16:1-256:1. In the present application, paclitaxel and tigecycline show significant synergistic effect, and the FICI is 0.1875-0.3125 (all less than 0.5), that is, the combination of the two shows significant synergistic effect. By using the combination of paclitaxel, the use amount of tigecycline can be effectively reduced, and the use amount of tigecycline alone is 1 to 16 times higher than that of the combination of paclitaxel and tigecycline.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology. Specifically, it relates to a novel use of paclitaxel as a potentiator for tigecycline. Background Technology

[0002] Tigecycline (TGC) is a new generation of antibiotics, belonging to the glycylcycline class. It was approved for marketing in the United States in 2005 and in my country in early 2012. Tigecycline's main mechanism of action is similar to other antibiotics: it reversibly binds to the helical region (H34) on the 30S subunit of the bacterial ribosome, preventing the entry of tRNA and inhibiting bacterial protein translation (i.e., peptide chain elongation), ultimately blocking bacterial protein synthesis and limiting bacterial growth. However, due to the addition of an N-alkyl-glycylamino side chain at the C9 position of the D ring, tigecycline's binding affinity to the ribosome is enhanced, broadening its antibacterial spectrum. Its ability to inhibit protein synthesis is 3 to 20 times greater than other classes of drugs. Tigecycline effectively overcomes the main mechanisms of antibiotic resistance, such as the specific efflux pump Tet(A) and the ribosomal protective protein Tet(M).

[0003] Paclitaxel (PTX), an anticancer drug, has the molecular formula C47H51NO14, a molecular weight of 853.91 g / mol, and a melting point of 213℃. It is a white crystalline powder, a tricyclic diterpenoid, and a taxane chemotherapy product belonging to the taxane class. It is naturally found in the bark and needles of the yew tree (Taxus breviscapus), and exhibits high efficacy, low toxicity, and broad-spectrum activity. The structural formula of paclitaxel is shown below:

[0004]

[0005] In recent years, with the increased use of tigecycline and the occurrence of other antibiotic abuse, the therapeutic effect of tigecycline has relatively declined, leading to tigecycline resistance. According to the EUCAST (European Union Standard for Drug Susceptibility Testing), bacteria exhibit resistance when the MIC (micronizable concentration) of tigecycline against Enterobacteriaceae is 4 μg / mL. Currently, tigecycline resistance is showing a significant upward trend. Therefore, reducing the occurrence of resistance peaks through potentiators has important clinical application significance. To date, there are no studies using paclitaxel as a potentiator for tigecycline. Summary of the Invention

[0006] The purpose of this invention is to provide a novel use of paclitaxel in enhancing the antibacterial activity of antimicrobial agents. In the preparation of compositions or compound formulations, paclitaxel is used to enhance the antibacterial activity of antimicrobial agents, wherein the amount of paclitaxel is 128 μg / mL, and the amount of tigecycline is 4 μg / mL or 8 μg / mL. Preferably, when the amount of paclitaxel is 128 μg / mL, the amount of tigecycline is 4-8 μg / mL.

[0007] A novel use of paclitaxel in enhancing the antibacterial activity of antimicrobial agents: In the preparation of compositions or compound formulations, paclitaxel is used to enhance the antibacterial activity of tigecycline, with a paclitaxel to tigecycline ratio of 16:1 or 32:1. Preferably, the paclitaxel-tigecycline ratio is between 16:1 and 32:1.

[0008] A novel use of paclitaxel in enhancing the antibacterial activity of antimicrobial agents: In the preparation of compositions or compound formulations, paclitaxel is used to enhance the antibacterial activity of tigecycline, with a paclitaxel-to-tigecycline ratio of 64:1, 128:1, or 256:1. Preferably, the paclitaxel-to-tigecycline ratio is between 64:1 and 256:1.

[0009] A novel use of paclitaxel in enhancing the antibacterial activity of antimicrobial agents: In the preparation of compositions or compound formulations, paclitaxel is used to enhance the antibacterial activity of tigecycline. When the amount of paclitaxel is 256 μg / mL, the amount of tigecycline is 1 μg / mL, or 2 μg / mL, or 4 μg / mL. Preferably, when the amount of paclitaxel is 256 μg / mL, the amount of tigecycline is 1-4 μg / mL.

[0010] A novel use of paclitaxel in enhancing the antibacterial activity of antimicrobial agents: In the preparation of compositions or compound formulations, paclitaxel is used to enhance the antibacterial activity of tigecycline, with the ratio of paclitaxel to tigecycline between 16:1 and 256:1.

[0011] Another object of this invention is to provide a novel use of paclitaxel in enhancing the antibacterial activity of tigecycline, wherein the antibacterial activity includes anti-Escherichia coli and Klebsiella pneumoniae. In the preparation of compositions or compound formulations, paclitaxel is used to enhance the antibacterial activity of the antibacterial agent, wherein the amount of paclitaxel is 128 μg / mL and the amount of tigecycline is 4 μg / mL or 8 μg / mL. Preferably, when the amount of paclitaxel is 128 μg / mL, the amount of tigecycline is 4-8 μg / mL. In the novel use of paclitaxel in enhancing the antibacterial activity of tigecycline against Escherichia coli and Klebsiella pneumoniae, in the preparation of compositions or compound formulations, paclitaxel is used to enhance the antibacterial activity of tigecycline, wherein the ratio of paclitaxel to tigecycline is 16:1 or 32:1. Preferably, when paclitaxel is used to enhance the antibacterial activity of tigecycline, the ratio of paclitaxel to tigecycline is between 16:1 and 32:1.

[0012] A novel use of paclitaxel in enhancing the antibacterial activity of tigecycline against *Escherichia coli* and *Klebsiella pneumoniae*: In the preparation of compositions or compound formulations, paclitaxel is used to enhance the antibacterial activity of tigecycline. When the amount of paclitaxel is 256 μg / mL, the amount of tigecycline is 1 μg / mL, 2 μg / mL, or 4 μg / mL. Preferably, when the amount of paclitaxel is 256 μg / mL, the amount of tigecycline is 1-4 μg / mL.

[0013] A novel use of paclitaxel in enhancing the antibacterial activity of tigecycline against Escherichia coli and Klebsiella pneumoniae: In the preparation of compositions or compound preparations, paclitaxel is used to enhance the antibacterial activity of tigecycline in a ratio of paclitaxel to tigecycline of 16:1, 32:1, 64:1, 128:1, or 256:1. Preferably, paclitaxel is used to enhance the antibacterial effect of tigecycline against Escherichia coli and Klebsiella pneumoniae, with a paclitaxel to tigecycline ratio between 64:1 and 256:1; preferably, the paclitaxel to tigecycline ratio is between 64:1 and 256:1; preferably, the paclitaxel to tigecycline ratio is between 64:1 and 128:1; preferably, the paclitaxel to tigecycline ratio is between 128:1 and 256:1; preferably, the paclitaxel to tigecycline ratio is between 16:1 and 32:1.

[0014] A novel use of paclitaxel in enhancing the antibacterial activity of tigecycline against Escherichia coli and Klebsiella pneumoniae: In the preparation of compositions or compound formulations, paclitaxel is used to enhance the antibacterial activity of tigecycline, with a ratio of paclitaxel to tigecycline between 16:1 and 256:1.

[0015] Preferably, paclitaxel is used to enhance the antibacterial effect of tigecycline, with tigecycline at a dosage of 4-8 μg / mL and paclitaxel at a dosage of 128 μg / mL. More preferably, a combination of tigecycline at a dosage of 1-4 μg / mL and paclitaxel at a dosage of 256 μg / mL is used.

[0016] Another object of this invention is to provide a novel use of paclitaxel in enhancing the antibacterial effect of tigecycline against Escherichia coli. In the preparation of compositions or compound formulations, paclitaxel is used to enhance the antibacterial effect of tigecycline against Escherichia coli, wherein the dosage of paclitaxel is 128 μg / mL, and the dosage of tigecycline is 4 μg / mL or 8 μg / mL. Preferably, when the dosage of paclitaxel is 128 μg / mL, the dosage of tigecycline is 4-8 μg / mL.

[0017] This invention provides a novel use of paclitaxel in enhancing the antibacterial effect of tigecycline against Escherichia coli-like bacteria. The antibacterial bacteria strains include strains ZZ9DT16R, ZC9CTR2, and JXCE34. Paclitaxel is used to enhance the antibacterial effect of tigecycline against E. coli strains ZZ9DT16R, ZC9CTR2, and JXCE34. The dosage of paclitaxel is 128 μg / mL, and the dosage of tigecycline is 4 μg / mL or 8 μg / mL. Preferably, when the dosage of paclitaxel is 128 μg / mL, the dosage of tigecycline is 4-8 μg / mL. Preferably, the ratio of paclitaxel to tigecycline is 16:1 or 32:1; preferably, the ratio of paclitaxel to tigecycline used to enhance the antibacterial effect of tigecycline is between 16:1 and 32:1.

[0018] A novel use of paclitaxel in enhancing the antibacterial activity of tigecycline against Escherichia coli: In the preparation of compositions or compound formulations, paclitaxel is used to enhance the antibacterial activity of tigecycline, with a paclitaxel-to-tigecycline ratio of 16:1 or 32:1. Preferably, the paclitaxel-to-tigecycline ratio is between 16:1 and 32:1.

[0019] A novel use of paclitaxel in enhancing the antibacterial activity of tigecycline against Escherichia coli: In the preparation of compositions or compound formulations, paclitaxel is used to enhance the antibacterial activity of tigecycline. When the amount of paclitaxel is 256 μg / mL, the amount of tigecycline is 1 μg / mL, 2 μg / mL, or 4 μg / mL. Preferably, when the amount of paclitaxel is 256 μg / mL, the amount of tigecycline is 1-4 μg / mL.

[0020] A novel use of paclitaxel in enhancing the antibacterial activity of tigecycline against *Escherichia coli*: In the preparation of compositions or compound formulations, paclitaxel is used to enhance the antibacterial activity of tigecycline, with a paclitaxel-to-tigecycline ratio of 64:1, 128:1, or 256:1. Preferably, the paclitaxel-to-tigecycline ratio is between 64:1 and 256:1. More preferably, the paclitaxel-to-tigecycline ratio is between 64:1 and 128:1. More preferably, the paclitaxel-to-tigecycline ratio is between 128:1 and 256:1.

[0021] Another object of the present invention is to provide a novel use of paclitaxel in enhancing the anti-Klebsiella pneumoniae effect of tigecycline. In the preparation of a composition or compound formulation, paclitaxel is used to enhance the anti-Klebsiella pneumoniae effect of tigecycline, wherein the amount of paclitaxel is 512 μg / mL and the amount of tigecycline is 4 μg / mL. Preferably, when the amount of paclitaxel is 128 μg / mL, the amount of tigecycline is 4 μg / mL.

[0022] A novel use of paclitaxel in enhancing the antibacterial activity of tigecycline against Klebsiella pneumoniae: In the preparation of compositions or compound formulations, paclitaxel is used to enhance the antibacterial activity of tigecycline, with a paclitaxel-to-tigecycline ratio of 32:1 or 64:1. Preferably, when paclitaxel is used to enhance the antibacterial activity of tigecycline, the paclitaxel-to-tigecycline ratio is between 32:1 and 64:1.

[0023] A novel use of paclitaxel in enhancing the antibacterial effect of tigecycline against Klebsiella pneumoniae: In the preparation of compositions or compound formulations, paclitaxel is used to enhance the antibacterial effect of tigecycline, wherein the amount of paclitaxel is 256 μg / mL and the amount of tigecycline is 4 μg / mL. Preferably, the amount of paclitaxel is 128 μg / mL and the amount of tigecycline is 4 μg / mL.

[0024] A novel use of paclitaxel in enhancing the antibacterial activity of tigecycline against Klebsiella pneumoniae: In the preparation of compositions or compound formulations, paclitaxel is used to enhance the antibacterial effect of tigecycline, with a paclitaxel-to-tigecycline ratio of 64:1 or 32:1. Preferably, when paclitaxel is used to enhance the antibacterial activity of tigecycline, the paclitaxel-to-tigecycline ratio is between 32:1 and 64:1.

[0025] Another object of the present invention is to provide the application of paclitaxel in the preparation of a synergist for the antibacterial activity of tigecycline, wherein the ratio of paclitaxel to tigecycline is 16:1, 32:1, or 64:1. Preferably, the ratio of paclitaxel to tigecycline is between 16:1 and 64:1; more preferably, the ratio is between 32:1 and 64:1.

[0026] The application of paclitaxel in the preparation of tigecycline as an antibacterial synergist, wherein the ratio of paclitaxel to tigecycline is 32:1, 64:1, 128:1 or 256:1; preferably, the ratio of paclitaxel to tigecycline is between 32:1 and 256:1.

[0027] Another object of the present invention is to provide the use of paclitaxel in the preparation of an antibacterial synergist for tigecycline against Escherichia coli and Klebsiella pneumoniae, wherein the ratio of paclitaxel to tigecycline is 16:1, 32:1, or 64:1. Preferably, the ratio of paclitaxel to tigecycline is between 16:1 and 64:1; more preferably, the ratio is between 32:1 and 64:1.

[0028] The application of paclitaxel in the preparation of an antibacterial synergist for tigecycline against *Escherichia coli* and *Klebsiella pneumoniae*, wherein the *Escherichia coli* strains are ZZ9DT16R, ZC9CTR2, and JXCE34, and the *Klebsiella pneumoniae* strain is HC139, wherein the ratio of paclitaxel to tigecycline is 32:1, 64:1, 128:1, or 256:1. Preferably, the ratio of paclitaxel to tigecycline is between 64:1 and 256:1. More preferably, the ratio of paclitaxel to tigecycline is between 64:1 and 128:1. More preferably, the ratio of paclitaxel to tigecycline is between 128:1 and 256:1. The application of paclitaxel in the preparation of an antibacterial synergist for tigecycline against *Escherichia coli* and *Klebsiella pneumoniae*, wherein the *Escherichia coli* strains are ZZ9DT16R, ZC9CTR2, and JXCE34, and the *Klebsiella pneumoniae* strain is HC139, wherein the ratio of paclitaxel to tigecycline is 16:1, 32:1, or 64:1. Preferably, the ratio of paclitaxel to tigecycline is between 16:1 and 64:1, or between 16:1 and 32:1; more preferably, the ratio is between 32:1 and 64:1.

[0029] The application of paclitaxel in the preparation of tigecycline as an antibacterial synergist against Escherichia coli and Klebsiella pneumoniae, wherein the ratio of paclitaxel to tigecycline is 16:1, 32:1, 64:1, 128:1 or 256:1; preferably, the ratio of paclitaxel to tigecycline is between 16:1 and 256:1; more preferably, the ratio of paclitaxel to tigecycline is between 32:1 and 256:1.

[0030] Another object of the present invention is to provide the application of paclitaxel in the preparation of an antibacterial synergist for tigecycline against Escherichia coli, wherein the ratio of paclitaxel to tigecycline is 16:1 or 32:1. Preferably, the ratio of paclitaxel to tigecycline is between 16:1 and 32:1. In the application of paclitaxel in the preparation of an antibacterial synergist for tigecycline against Escherichia coli, the Escherichia coli strains include strains ZZ9DT16R, ZC9CTR2, and JXCE34, wherein the ratio of paclitaxel to tigecycline is 64:1, 128:1, or 256:1. Preferably, the ratio of paclitaxel to tigecycline is between 64:1 and 256:1. Preferably, the ratio of paclitaxel to tigecycline is between 64:1 and 128:1. Preferably, the ratio of paclitaxel to tigecycline is between 128:1 and 256:1.

[0031] Another object of the present invention is to provide the use of paclitaxel in the preparation of an antibacterial potentiator of tigecycline against Klebsiella pneumoniae, wherein the ratio of paclitaxel to tigecycline is 32:1 or 64:1; preferably, the ratio of paclitaxel to tigecycline is between 32:1 and 64:1. The Klebsiella pneumoniae strain includes strain HC139, wherein the ratio of paclitaxel to tigecycline is 32:1 or 64:1; preferably, the ratio of paclitaxel to tigecycline is between 32:1 and 64:1.

[0032] Another object of the present invention is to provide a paclitaxel antibacterial composition comprising paclitaxel and tigecycline, wherein the paclitaxel antibacterial composition is used against *Escherichia coli* or *Klebsiella pneumoniae*, and wherein the ratio of paclitaxel to tigecycline is 16:1, 32:1, or 256:1. Preferably, the ratio of paclitaxel to tigecycline in the antibacterial composition is between 16:1 and 256:1; more preferably, the ratio of paclitaxel to tigecycline in the antibacterial composition is between 32:1 and 256:1.

[0033] Another object of the present invention is to provide a paclitaxel antibacterial composition comprising paclitaxel and tigecycline, wherein the paclitaxel antibacterial composition is used against *Escherichia coli* or *Klebsiella pneumoniae*, and wherein the ratio of paclitaxel to tigecycline is 32:1, 64:1, 128:1, or 256:1. Preferably, the ratio of paclitaxel to tigecycline is between 64:1 and 256:1. Preferably, the ratio of paclitaxel to tigecycline is between 64:1 and 128:1. Preferably, the ratio of paclitaxel to tigecycline is between 128:1 and 256:1.

[0034] Another object of the present invention is to provide a paclitaxel antibacterial combination preparation, comprising paclitaxel and tigecycline, for use against Escherichia coli or Klebsiella pneumoniae, wherein the ratio of paclitaxel to tigecycline is 16:1, 32:1, 64:1, 128:1, or 256:1. Preferably, the ratio of paclitaxel to tigecycline is any ratio between 16:1 and 256:1.

[0035] Another object of the present invention is to provide a paclitaxel antibacterial combination preparation, comprising paclitaxel and tigecycline, for use against Escherichia coli or Klebsiella pneumoniae, wherein the ratio of paclitaxel to tigecycline is 16:1 or 32:1. Preferably, the ratio of paclitaxel to tigecycline is any ratio between 16:1 and 32:1.

[0036] Another object of the present invention is to provide an antibacterial test method, or a target analyte test method, for the use of paclitaxel in enhancing tigecycline against Escherichia coli, the method comprising the following steps:

[0037] S1: Dissolve paclitaxel and tigecycline in a solvent;

[0038] S2: Paclitaxel and tigecycline are mixed in a certain proportion to obtain a composition;

[0039] S3: Use the composition from step S2 for MIC testing, FICI testing, time-sterilization curve testing, or target analyte testing.

[0040] The solvent mentioned in step S1 includes dimethyl sulfoxide or deionized water.

[0041] In step S2, the ratio of paclitaxel to tigecycline is 16:1. Preferably, the ratio of paclitaxel to tigecycline is 16:1.

[0042] When the dosage of paclitaxel in step S3 is 128 μg / mL or 256 μg / mL, the dosage of tigecycline is 1-8 μg / mL. Preferably, when the dosage of paclitaxel is 256 μg / mL, the dosage of tigecycline is 1 μg / mL, 2 μg / mL, or 4 μg / mL. Preferably, when the dosage of paclitaxel is 256 μg / mL, the dosage of tigecycline is 1-4 μg / mL.

[0043] In this article, the term "synergistic effect" refers to the effect of two drugs combined being greater than the algebraic sum of the effects of the two drugs alone.

[0044] In this article, the term "additive effect" means that the combined effect of two drugs is equal to the algebraic sum of their individual effects.

[0045] In this article, the term "antagonistic effect" refers to the combined effect of two drugs being less than the sum of their individual effects.

[0046] In this article, the term "anti-Escherichia coli effect" refers to the inhibition of the proliferation or growth of Escherichia coli, or the killing of Escherichia coli.

[0047] In this article, the term "anti-Klebsiella pneumoniae effect" refers to the inhibition of the proliferation or growth of Klebsiella pneumoniae, or the killing of Klebsiella pneumoniae.

[0048] The present invention has the following beneficial effects: 1. When the dosage of paclitaxel is 128 μg / mL or 256 μg / mL, the dosage of tigecycline is 1-8 μg / mL, which can significantly enhance the antibacterial activity of tigecycline against Escherichia coli. 2. The combined use of paclitaxel and tigecycline both show significant synergistic effects, with a FICI of 0.1875-0.3125 (all less than 0.5), indicating a significant synergistic effect. 3. By using paclitaxel in combination, the dosage of tigecycline can be effectively reduced. The dosage of tigecycline used alone is 1 to 16 times higher than that of paclitaxel used in combination, while synergistically improving bacterial resistance to tigecycline. 4. The combined use of paclitaxel and tigecycline can synergistically reverse drug resistance, with a FICI of 0.1875-0.3125. The combined use kills all bacteria after 12 hours. Attached Figure Description

[0049] Figure 1 The time-kill curves of paclitaxel and tigecycline on strain ZZ9DT16R are shown. Detailed Implementation

[0050] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0051] Example 1

[0052] Materials and methods

[0053] 1. Drug testing

[0054] Paclitaxel (PTX): Shanghai Maclean Biochemical Technology Co., Ltd., 98% purity.

[0055] Tigecycline (TGC): Shanghai Yuanye Biotechnology Co., Ltd., 98% purity.

[0056] Paclitaxel was prepared to a concentration of 5120 μg / mL using dimethyl sulfoxide. Tigecycline was prepared to a concentration of 1280 μg / mL using deionized water. The test drugs were stored at -20°C. Before the test, the drug stock solution was removed and allowed to thaw at room temperature, thoroughly mixed, and then pharmacodynamic tests were performed.

[0057] 2. Strains

[0058] The clinically isolated *Escherichia coli* strains were *E. coli* ZZ9DT16R, ZC9CTR2, and JX21CE34, and the clinically isolated *Klebsiella pneumoniae* strains were *Klebsiella pneumoniae* HC139, provided by the Pharmacology Laboratory of Henan Agricultural University. *E. coli* and *Klebsiella pneumoniae* were activated by streaking on MacConkey agar, incubated at 37°C for 16–18 h, and stored at 4°C for later use.

[0059] 3. Culture medium

[0060] LB broth medium: Produced by Beijing Aoboxing Biotechnology Co., Ltd. Prepare 1000 ml according to the product instructions, autoclave at 121℃ for 15 minutes, and store at 4℃ for later use. SS agar medium: Produced by Beijing Aoboxing Biotechnology Co., Ltd. Prepare 1000 ml according to the product instructions, heat to boiling to fully dissolve and sterilize, cool to 60℃, pour into sterile Petri dishes, and store at 4℃ for later use. MHB broth medium: Produced by Beijing Aoboxing Biotechnology Co., Ltd. Prepare 1000 ml according to the product instructions, autoclave at 121℃ for 15 minutes, and store at 4℃ for later use.

[0061] 4. Instruments

[0062] High-pressure steam sterilizer (Shanghai Shenan Medical Instrument Factory); AB204-N electronic analytical balance (Mettler-Tolly Instruments Shanghai Co., Ltd.); HZQ-R air bath shaker (Beijing Liuyi Instrument Factory); DH-600 electric thermostatic incubator (Beijing Kewei Yongxing Instrument Co., Ltd.); DHG-9140A vacuum drying oven (Shanghai Yiheng Experimental Instrument Co., Ltd.); SW-CJ-JC clean bench (Suzhou Antai Air Technology Co., Ltd.).

[0063] 5. Preparation of bacterial culture

[0064] Single colonies of *Escherichia coli* or *Klebsiella pneumoniae* were picked from MacConkey agar stored at 4°C and inoculated into 5 mL of LB broth. The culture was then incubated at 37°C with shaking at 180 rpm to induce the bacteria to enter the late exponential growth phase. The bacterial culture was then transferred to MHB broth to dilute the original bacterial concentration to 10-1. 5 CFU / mL. Add the appropriate concentration of paclitaxel to the bacterial suspension for later use. The final concentrations of paclitaxel in the bacterial suspensions were 128 μg / mL and 256 μg / mL, respectively.

[0065] 6. Preparation of Antimicrobial Sensitivity Plates

[0066] Drug susceptibility testing was performed using the bacterial suspension from step 5. A sterile 96-well reaction plate was used. In well 1 of the first row, 126 μL of freshly prepared paclitaxel-containing bacterial suspension and 14 μL of 1280 μg / mL tigecycline were added; in wells 2-12, 70 μL of freshly prepared paclitaxel-containing bacterial suspension was added; in wells 1-7 of the second row, 70 μL of freshly prepared paclitaxel-containing bacterial suspension was also added; and in well 8, 70 μL of MHB broth was added as a blank control. Serial dilutions were performed on wells 1-12 in the first row and wells 1-6 in the second row to achieve final tigecycline concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125, and 0.00390625 μg / mL, respectively. Well 7 in the second row contained a drug-free MHB bacterial suspension as a negative control, and well 8 contained a drug-free bacterial culture as a positive control. The susceptibility testing plates were incubated at 37°C for 16-20 hours.

[0067] 7. Determination of MIC value

[0068] After incubating the 96-well plates containing bacteria at 37°C for 16-20 hours, the MIC (minimum inhibitory concentration) results were read. The MIC value (minimum inhibitory concentration) of the drug is the drug concentration in the preceding clear well in the 96-well plate. All experiments were performed in triplicate. Only when the MIC values ​​could be accurately reproduced was the experiment accepted; if the MIC values ​​differed by more than one concentration, a new experiment was required until the requirements were met. The results are shown in Table 1.

[0069] Table 1. MICs of paclitaxel combined with tigecycline against tested bacterial strains

[0070]

[0071] Note: 1 / 2MIC paclitaxel: paclitaxel added at a concentration of 256 μg / mL; 1 / 4MIC paclitaxel: paclitaxel added at a concentration of 128 μg / mL;

[0072] The results showed that the MIC values ​​of tigecycline against strains ZZ9DT16R, ZC9CTR2, JXCE34, and HC139 were 16, 4, 32, and 16 μg / mL, respectively. The MIC value of paclitaxel against strains ZZ9DT16R, ZC9CTR2, JXCE34, and HC139 was 512 μg / mL. When 1 / 2 MIC (256 μg / mL) and 1 / 4 MIC (128 μg / mL) of paclitaxel were added, the MIC values ​​of tigecycline against ZZ9DT16R were 4 and 8 μg / mL, respectively; against ZC9CTR2 were 1 and 4 μg / mL, respectively; against JXCE34 were 2 and 4 μg / mL, respectively; and against HC139 were 4 and 4 μg / mL, respectively.

[0073] Compared with the MIC values ​​when tigecycline was used alone, when 256 μg / mL and 128 μg / mL of paclitaxel were added, the MIC of tigecycline against strain ZZ9DT16R decreased by 4 and 2 times, respectively; the MIC of tigecycline against strain ZC9CTR2 decreased by 4 and 1 times, respectively; the MIC of tigecycline against strain JXCE34 decreased by 16 and 8 times, respectively; and the MIC of tigecycline against strain HC139 decreased by 4 times.

[0074] The results showed that when paclitaxel was used to enhance the antibacterial effect of tigecycline, the dosage of paclitaxel to tigecycline was reduced by 1 to 16 times compared with the dosage of tigecycline alone when the ratio of paclitaxel to tigecycline was 16:1, 32:1, 64:1, 128:1 or 256:1.

[0075] The results showed that paclitaxel concentrations of 256 μg / mL and 128 μg / mL significantly enhanced the antibacterial activity of tigecycline against Escherichia coli ZZ9DT16R, ZC9CTR2, JXCE34, and Klebsiella pneumoniae HC139.

[0076] Example 2

[0077] Materials and methods

[0078] 1. Drug testing:

[0079] Same as Example 1.

[0080] 2. Strains

[0081] Same as Example 1.

[0082] 3. Culture medium

[0083] Same as Example 1.

[0084] 4. Instruments

[0085] Same as Example 1.

[0086] 5. Preparation of bacterial culture

[0087] Same as Example 1.

[0088] 6. Preparation of Antimicrobial Sensitivity Plates

[0089] Based on the microbroth dilution method, we obtained the MIC values ​​of PTX and TGC as single drugs, and selected serial dilutions of PTX and TGC. Specifically, 100 μL of MHB was allocated to 8×8 96-well plates, and then TGC and PTX were diluted 2-fold along the x-axis and y-axis, respectively. MIC values ​​for PTX and TGC as single drugs, as well as negative and positive controls, were calculated. (The 96-well plates were placed in a constant temperature incubator at 37℃ for 16–18 h, and the results were observed and recorded. The experiment was repeated three times.)

[0090] The partial inhibitory concentration index (FICI) was used as the criterion for evaluating the checkerboard susceptibility testing. The FICI values ​​of compounds A and B were defined by the following formula:

[0091] FICI = FIC A +FIC B =MIC AB / MIC A +MIC BA / MIC B .

[0092] MIC A It is the MIC of compound A alone, MIC AB It is the MIC when compound A is combined. B It is the MIC of compound B. BA The FICI values ​​represent the MICs of compound B in combination. FICI < 0.5 indicates a synergistic effect; 0.5 ≤ FICI ≤ 1 indicates an additive effect; 1 < FICI ≤ 2 indicates an indifferent effect; and FICI > 2 indicates an antagonistic effect. The results are shown in Table 2.

[0093] Table 2. Combined antibacterial effects of paclitaxel and tigecycline

[0094]

[0095] The results showed that paclitaxel alone had a weak inhibitory effect on the tested strains, but when used in combination with TGC, it showed a synergistic effect on TGC. The FICI values ​​were 0.1875–0.3125, all <0.5, indicating that the combination of the two showed a synergistic effect.

[0096] Example 3

[0097] Materials and methods

[0098] 1. Drug testing:

[0099] Same as Example 1.

[0100] 2. Strains

[0101] Same as Example 1.

[0102] 3. Culture medium

[0103] Same as Example 1.

[0104] 4. Instruments

[0105] Same as Example 1.

[0106] 5. Preparation of bacterial culture

[0107] Same as Example 1.

[0108] 6. Time-sterilization curve

[0109] Escherichia coli ZZ9DT16R was selected for time-kill curve testing. Single colonies were inoculated into fresh LB broth and incubated overnight at 37°C and 180 rpm. The bacterial culture was then transferred 1:100 to 10 mL of fresh LB broth, resulting in 5 experimental groups.

[0110] Group A: Control;

[0111] Group B: 1 / 2 MIC TGC;

[0112] Group C: 1 / 2 MIC PTX;

[0113] Group D: 1 / 2MIC PTX + 1 / 2MIC TGC;

[0114] Group E: 1 / 4MIC PTX + 1 / 2MIC TGC.

[0115] After grouping, the cells were incubated at 37℃ with shaking at 180 rpm. At 0h, 2h, 4h, 8h, 12h, and 24h, 100 μL of bacterial suspension was taken and serially diluted 10-fold with PBS buffer. 100 μL of each diluted suspension was then spread onto LB agar plates and incubated at 37℃ for 16–18h. Finally, the colonies on the agar plates were counted, and the result was plotted with time on the x-axis. 10 Plot a time-sterilization curve with CFU / mL as the ordinate. The experiment was repeated three times.

[0116] Result interpretation: At 24 hours, compared with the single-drug group, the colony count in the combination therapy group was reduced by ≥2 log. 10 Defined as a synergistic effect. The results are as follows: Figure 1 As shown.

[0117] Experimental results showed that with 1 / 2 MIC PTX alone, the bacterial growth was slow in the first 12 hours, indicating a weak inhibitory effect of PTX. The bacterial count decreased at 24 hours, indicating a weak bactericidal effect. 1 / 2 MIC TGC initially showed some bactericidal effect, but the bacterial count increased rapidly between 4 and 24 hours. With 1 / 2 MIC TGC + 1 / 4 MIC PTX, a significant bactericidal effect was observed from 2 to 24 hours, with a reduction in colony count greater than 2 log₂. 10 CFU / mL. The 1 / 2MIC TGC + 1 / 2MIC PTX combination showed a more significant bactericidal effect, completely killing bacteria within 12 hours. The time-bacterial control curve results were largely consistent with the combined drug susceptibility results, indicating that PTX alone had a weak inhibitory effect. The combination of 1 / 2MIC PTX or 1 / 4MIC PTX with TGC significantly improved the antibacterial activity of TGC against drug-resistant bacteria, demonstrating a good synergistic effect in reversing drug resistance.

[0118] The above experiments showed that paclitaxel concentrations of 128 μg / mL and 256 μg / mL significantly enhanced the antibacterial activity of tigecycline against *Escherichia coli* ZZ9DT16R, ZC9CTR2, JX21CE34, and *Klebsiella pneumoniae* HC139. The combination of paclitaxel and tigecycline showed significant synergistic effects, with FICI values ​​ranging from 0.1875 to 0.3125, all < 0.5, indicating a significant synergistic effect. Time-kill curves showed that the combined treatment exhibited significant bactericidal activity. Specifically, 1 / 2 MIC PTX + 1 / 2 MIC TGC completely killed bacteria within 12 hours, consistent with the combined drug susceptibility results, indicating that PTX alone had a weak antibacterial effect. The combination of 1 / 2 MIC PTX or 1 / 4 MIC PTX with TGC significantly improved the antibacterial activity of TGC against drug-resistant bacteria, demonstrating a good synergistic effect in reversing drug resistance.

[0119] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of paclitaxel in the preparation of an antibacterial synergist for tigecycline against Escherichia coli and Klebsiella pneumoniae, characterized in that, The ratio of paclitaxel to tigecycline is between 32:1 and 256:

1.

2. The application according to claim 1, characterized in that, The ratio of paclitaxel to tigecycline was 32:

1.

3. The application according to claim 1, characterized in that, The ratio of paclitaxel to tigecycline is 64:1, 128:1, or 256:

1.

4. The application according to claim 1, characterized in that, The ratio of paclitaxel to tigecycline is between 64:1 and 256:1.

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