Application of a zinc ion chelator in the treatment of Candida albicans biofilm with azole drugs

Abstract

The present invention discloses a method for enhancing the anti-Candida albicans biofilm activity of azole drugs by utilizing a zinc ion chelator. Through the combined action of the zinc ion chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and fluconazole, the activity of the Candida albicans biofilm can be reduced, the early adhesion process, the mid-term yeast to hyphae conversion process, and the late maturation process of the Candida albicans biofilm formation can be inhibited, the Candida albicans biofilm structure can be destroyed, and the high drug resistance of the Candida albicans biofilm to fluconazole can be reversed, and its pathogenicity can be reduced. The method is expected to solve the problems of high drug resistance and persistent infection caused by the formation of the Candida albicans biofilm, and provide a direction for the treatment of clinical fungal infections and drug research and development.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of TPEN in treating Candida albicans biofilm with fluconazole. Background Art

[0002] Candida albicans is a fungus that is widely present in nature and in the human body. Its infection is often associated with a decrease in human immunity. Up to 80% of infections are related to the biofilm it forms. This biofilm is a complex three-dimensional structure composed of yeast spores, pseudohyphae and fungal hyphae, as well as extracellular matrix. It exhibits extremely strong resistance and tolerance to the host immune defense system and environmental stress factors. Compared with the planktonic Candida albicans, the formation of Candida albicans biofilm leads to a more than 1,000-fold increase in resistance to antifungal drugs represented by fluconazole, resulting in failure of clinical antifungal treatment. Therefore, there is an urgent need to develop new methods to overcome the azole resistance of Candida albicans caused by biofilm formation.

[0003] N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) is a zinc ion chelating agent with the chemical formula Because it is lipid-soluble, it can permeate cell membranes and chelate intracellular zinc ions. In the biomedical field, TPEN is often used to study the role of zinc ions within cells and in various biological processes. For example, it investigates the structure and function of biomacromolecules such as zinc metalloproteinases and chelates zinc ions within bacterial cells to produce antibacterial effects. To date, there are no reports on the use of TPEN in Candida albicans biofilms, nor is there any evidence that TPEN has a substantial effect on fluconazole's antibacterial effects against Candida albicans biofilms. Summary of the Invention

[0004] The purpose of the present invention is to overcome at least one deficiency of the prior art and provide an application of a zinc ion chelator in the treatment of Candida albicans biofilm with azole drugs.

[0005] The technical solution adopted by the present invention is:

[0006] The present invention provides a method for enhancing the anti-Candida albicans biofilm activity of fluconazole (FLC) by using the zinc chelator TPEN, aiming to reduce the activity of the Candida albicans biofilm, inhibit the early adhesion process, the mid-stage yeast to hyphae transformation process, and the late maturation process of the Candida albicans biofilm growth, and destroy the Candida albicans biofilm structure.

[0007] In a first aspect, the present invention provides an application of a zinc ion chelator in the preparation of an azole drug synergist for inhibiting Candida albicans biofilm, wherein the zinc ion chelator is N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.

[0008] In a second aspect, the present invention provides a use of a zinc ion chelator in the preparation of a drug synergist against Candida albicans biofilm, wherein the zinc ion chelator is N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.

[0009] In a third aspect, a zinc ion chelator is used in synergistically inhibiting Candida albicans biofilm with azole drugs, wherein the zinc ion chelator is N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.

[0010] In some examples, the azole drug is selected from fluconazole.

[0011] In some examples, the zinc ion chelator further includes N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine or a pharmaceutically acceptable solvate thereof.

[0012] In some examples, the drug is administered topically, orally, by injection, implant, rectum, spray, or inhalation.

[0013] In some examples, the dosage form of the drug includes but is not limited to solution, tincture, spirit, lotion, ointment, plaster, paste, oil, film, liniment, injection, tablet, granule, capsule, pill, sustained-release agent, oral liquid preparation, powder or gel.

[0014] In some examples, the drug further includes a pharmaceutical carrier or a pharmaceutically acceptable excipient.

[0015] In a fourth aspect, the present invention provides a composition for inhibiting Candida albicans or Candida albicans biofilm, wherein the effective components of the composition are N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine or a pharmaceutically acceptable solvate thereof and an azole drug.

[0016] The beneficial effects of the present invention are:

[0017] 1) TPEN and Fluconazole Combination Therapy: This invention proposes a novel combination therapy, combining TPEN and fluconazole, to provide a more effective approach for preventing and treating Candida albicans biofilm infections. This combination therapy is expected to disrupt the physiological functions of Candida albicans through TPEN's specific chelation of zinc ions, while fluconazole simultaneously exerts its antifungal effects, achieving a synergistic effect on fungal cells.

[0018] 2) Therapeutic mechanism targeting zinc ion dependence: Given the key role of zinc ions in Candida albicans, the solution of the present invention specifically targets this biological characteristic. By chelating zinc ions in cells with TPEN, it interferes with the transcriptional regulation and enzyme activity of the fungus, thereby weakening the formation and maintenance of biofilms, providing a new approach for the treatment of drug-resistant biofilms.

[0019] 3) Reduced risk of drug resistance: The present invention is expected to reduce the development of drug resistance. Through the dual mechanism of action of TPEN and fluconazole, the possibility of fungi developing resistance to a single drug is reduced, providing a long-term, sustainable treatment option for clinical treatment.

[0020] 4) Reduce toxic and side effects: The present invention aims to reduce the toxic and side effects that may be caused by the use of a single drug, provide a safer treatment plan, and improve the patient's treatment experience and treatment effect.

[0021] 5) Possibility of clinical application: The treatment regimen of the present invention is expected to be widely applicable in the clinical treatment of fluconazole-resistant Candida albicans biofilm infections, and has important public health significance and market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing the effect of TPEN concentration-dependently reducing the zinc ion concentration in Candida albicans cells in Example 1.

[0023] Figure 2 This is a graph showing the concentration-dependent inhibition of planktonic Candida albicans growth in Example 2.

[0024] Figure 3 TPEN enhances the antibacterial effect of fluconazole against planktonic Candida albicans in Example 3; wherein, A is a graph showing the growth inhibition effect of TPEN combined with fluconazole on Candida albicans in YPD medium; B is a graph showing the antibacterial effect of Candida albicans co-incubated with TPEN and fluconazole in RPMI 1640 medium for 24 hours, observed under a microscope; C is a graph showing the change in fluconazole sensitivity of Candida albicans detected by the disc diffusion method; and D is a statistical graph showing the diameter of the inhibition zone by the disc diffusion method.

[0025] Figure 4 This figure shows the inhibitory effect of TPEN on enhancing the adhesion of fluconazole to Candida albicans in the early stage of biofilm formation in Example 4.

[0026] Figure 5 This is a statistical diagram of the biomass of Candida albicans in the early stage of biofilm formation when TPEN enhances the inhibition of fluconazole on adhesion.

[0027] Figure 6 This figure shows the inhibitory effect of TPEN on the yeast-hyphae conversion of Candida albicans at the middle stage of biofilm formation in Example 5.

[0028] Figure 7 This figure shows the inhibitory effect of TPEN on Candida albicans biofilm maturation at the late stage of biofilm formation in Example 6.

[0029] Figure 8 This is a statistical diagram of the biomass of Candida albicans biofilms in Example 6, showing that TPEN enhances the inhibition of fluconazole on the maturation of biofilms in the late stage of biofilm formation.

[0030] Figure 9 This is a diagram showing the therapeutic effect of greater wax moth systemically infected with Candida albicans biofilm in Example 8. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited thereto.

[0032] The sources of experimental materials for each embodiment of the present invention are as follows:

[0033] 1. Drugs and Reagents

[0034]

[0035]

[0036] 2. Instruments

[0037]

[0038] 3. Experimental strains

[0039] FLC-sensitive Candida albicans SC 5314, a common strain in international laboratories, was purchased from Hangzhou Fenghai Biotechnology Co., Ltd.

[0040] 4. Experimental Animals

[0041] Six-week-old greater wax moth larvae were purchased from Tianjin Huiyude Biotechnology Co., Ltd., with a length of 2-3 cm, a weight of 250-350 mg, good vitality, and no black spots.

[0042] Example 1 Determination of zinc ion levels in Candida albicans cells

[0043] Experimental process:

[0044] 1) Weigh 5 mg of zinc ion fluorescent probe Zinquin ethyl ester powder into a 1.5 mL centrifuge tube and dissolve in 1.206 mL of DMSO to create a 10 mM stock solution of Zinquin ethyl ester. Filter the stock solution three times with a 0.22 μm syringe filter before use. Dilute the Zinquin ethyl ester stock solution with DMSO to a 40 μM working solution.

[0045] 2) Add 1×10 7cell / ml Candida albicans, and RPMI 1640 medium without drugs was used as a control.

[0046] 3) Incubate the suspension at 37°C with shaking at 200 rpm for 16 h. Centrifuge the suspension for 10 min (3000 rpm, 4°C), discard the supernatant, and centrifuge again. Repeat this process three times.

[0047] 4) Resuspend the cells in 40 μM Zinquin ethyl ester working staining solution and incubate at 37°C in the dark for 30 minutes. Centrifuge the suspension for 10 minutes (3000 rpm, 4°C), discard the supernatant, add physiological saline, vortex to mix, and centrifuge again. Repeat this process three times to remove any residual staining solution.

[0048] 5) Resuspend Candida albicans in physiological saline and transfer 200 μL of the suspension to a 96-well plate. Calibrate the plates to the same OD600 value. Use a microplate reader with an excitation wavelength of 355 nm and an emission wavelength of 491 nm to read the fluorescence intensity of each experimental group.

[0049] Experimental results: Figure 1 As shown in the figure, with the increase of TPEN concentration, the fluorescence intensity of zinc ions in Candida albicans cells gradually decreased, and the difference was statistically significant compared with the control group (**P<0.01), indicating that TPEN reduces the zinc ion level in Candida albicans cells by chelating zinc ions.

[0050] Example 2: Inhibitory effect of TPEN on the growth of planktonic Candida albicans

[0051] Experimental process:

[0052] 1) 1.0×10 6 cell·mL -1 Candida albicans was incubated in 5 mL of YPD liquid medium containing 20 μM, 100 μM, or 500 μM TPEN and cultured at 37° C. with continuous shaking (200 rpm). YPD liquid medium without drugs was used as a positive control.

[0053] 2) After the samples were thoroughly mixed at fixed time points (0, 1, 3, 6, 12, and 24 h), 100 μL of bacterial suspension was taken from each group, diluted in saline, and spread on YPD agar plates.

[0054] 3) Incubate at 37°C for 24–48 h and count the colony-forming units (CFU).

[0055] Experimental results: Figure 2As shown, in yeast amplification YPD liquid medium, as the concentration of TPEN increased, the growth of Candida albicans was significantly inhibited, indicating that TPEN inhibited the growth of planktonic Candida albicans in a concentration-dependent manner.

[0056] Example 3: Changes in Fluconazole Sensitivity of TPEN to Planktonic Candida albicans

[0057] Experimental process:

[0058] 1. Determination of MIC and MFC of Drugs against Candida albicans

[0059] 1) Take a 96-well plate and add 100 μL RPMI 1640 liquid medium and 5×10 3 CFU·mL -1 As a positive control, 100 μL of fluconazole working solution of 10 different concentrations were added to wells 2-11 in descending order of concentration, and 5×10 3 CFU·mL -1 The final concentrations of fluconazole in each well were 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg mL -1 The final concentration of the bacterial solution was 2.5x10 3 CFU·mL -1 Add 200 μL of RPMI 1640 liquid medium to well 12 as a negative control. After incubation at 37°C for 24-48 hours, measure the absorbance of each well at 600 nm. The minimum inhibitory concentration (MIC) of fluconazole is the lowest concentration that inhibits fungal growth by at least 80% compared to the positive control well.

[0060] 2) The MIC of TPEN was determined using the above method.

[0061] 3) After supplementing each well plate with TPEN at a final concentration of 0.5× MIC, the change in fluconazole MIC was determined using the above method.

[0062] 4) After incubating the 96-well plate for 48 hours, 100 μL of the solution from each well was spread on YPD solid medium and cultured at 37°C for 24-48 hours. The minimum fungicidal concentration (MFC) was the lowest drug concentration at which no colonies grew.

[0063] 5) The growth of Candida albicans after 36 h of drug incubation was observed under an inverted microscope, and images were acquired using cellSensDimension software (Olympus).

[0064] 2. Evaluation of Fluconazole Susceptibility Changes of Candida albicans by TPEN Using the Drug Sensitivity Paper Diffusion Method

[0065] 1) Add 5×10 6 CFU·mL -1 Candida albicans was cultured at 37°C with shaking at 200 rpm for 2 h.

[0066] 2) The bacterial suspension was inoculated onto a Mueller Hinton (MH) agar plate (containing 20 g / L glucose and 0.5 μg / mL methylene blue). A drug-susceptibility paper strip containing 25 μg of fluconazole was placed in the center of the plate. After incubation at 37°C for 24 h, the diameter of the inhibition zone (mm) was measured using an electronic vernier caliper. The image of the culture dish was photographed and recorded. The experiment was repeated three times.

[0067] Experimental results: As shown in Table 1, the MIC of fluconazole was 0.5 μg / ml and the MFC was >1024 μg / ml. After supplementation with 0.5×MICTPEN, the MIC of fluconazole decreased to 0.25 μg / ml and the MFC decreased to 128 μg / ml. Figure 3 As shown in Figure A, 1024 μg / ml fluconazole inhibited the growth of Candida albicans. The addition of 20 μM TPEN increased the growth inhibition of fluconazole. The addition of 100 μM TPEN resulted in a bactericidal effect of fluconazole within 24 h. Figure 3 As shown in Figure B, the growth of Candida albicans was observed under a microscope. Fluconazole limited the hyphal extension and colony formation of Candida albicans in a concentration-dependent manner. When TPEN was used in combination with fluconazole, the growth of Candida albicans was significantly inhibited, and hyphal extension and colony formation were further reduced. Figure 3 As shown in Figures C and 3D, the addition of TPEN significantly increased the diameter of the fluconazole inhibition zone of Candida albicans, and the difference was statistically significant (*P < 0.05). These results indicate that TPEN enhances the fluconazole sensitivity of planktonic Candida albicans.

[0068] Table 1 MICs and MFCs of drugs against Candida albicans

[0069]

[0070] Example 4 Effect of TPEN on Fluconazole Inhibition of Early Adhesion of Candida albicans Biofilm

[0071] Experimental process:

[0072] 1) 1×10 6 CFU / mL of Candida albicans were resuspended in 1 ml of RPMI 1640 medium containing drugs, and medium without drugs was used as a control, and added to 24-well plates and incubated for 2 h.

[0073] 2) Remove non-adherent cells with physiological saline, fix with 1 ml / well methanol, stain with 0.1% crystal violet, rinse with double-distilled water, dry at room temperature, and observe under an inverted microscope.

[0074] 3) Add 95% ethanol at 1 mL / well and incubate for 10 min to completely dissolve the crystal violet. Pipette 200 μl of the ethanol solution containing the crystal violet into another 96-well plate. Read the A570 value with a microplate reader to quantitatively analyze the biomass of the Candida albicans biofilm.

[0075] Experimental results: Figure 4 and Figure 5 As shown, in the early stages of biofilm formation, 1024 μg / ml fluconazole had no significant inhibitory effect on C. albicans adhesion, while 80 μM TPEN significantly inhibited C. albicans adhesion. The combined inhibitory effect of TPEN on C. albicans adhesion was superior to that of either drug alone, with a statistically significant difference (***P < 0.001). These results indicate that TPEN enhances the inhibitory effect of fluconazole on C. albicans adhesion in the early stages of biofilm formation.

[0076] Example 5 Effect of TPEN on Fluconazole Inhibition of Yeast-Hypha Transition in Mid-Stage Candida albicans Biofilm

[0077] Experimental process:

[0078] 1) Prepare RPMI 1640 culture medium (containing 10% fetal bovine serum) containing different concentrations of drugs, and medium without drugs as a control, add a final concentration of 1×10 6 CFU / mL of Candida albicans was cultured at 37 °C with shaking at 200 rpm for 4 h to induce hyphae formation of Candida albicans.

[0079] 2) 1 ml of bacterial suspension was added to a 24-well plate from each group and observed under an inverted microscope. Images were acquired using cellSens Dimension software (Olympus). Grayscale conversion, brightness, and contrast were adjusted using Adobe Photoshop 25.0.0 (Adobe Systems, California, USA).

[0080] Experimental results: Figure 6 As shown, both fluconazole and TPEN inhibited the budding, hyphae formation and elongation of Candida albicans in a concentration-dependent manner, and the combination of TPEN and fluconazole further enhanced the inhibitory effect on the yeast-hyphae conversion of Candida albicans.

[0081] Example 6 The role of TPEN in fluconazole inhibition of late maturation of Candida albicans biofilm

[0082] Experimental process:

[0083] 1) Adjust the concentration of Candida albicans to 1×10 in RPMI 1640 medium. 6 CFU / mL, add 1 ml of bacterial suspension to each well of a 24-well plate and incubate at 37°C for 2 h.

[0084] 2) Wash the non-adherent cells with physiological saline, and add 1 ml of RPMI 1640 medium containing drugs to each well. Medium without drugs serves as a control.

[0085] 3) After incubation at 37°C for 24 h, remove non-adherent cells with physiological saline, fix with 1 ml / well methanol, stain with 0.1% crystal violet, rinse with double-distilled water, dry at room temperature, and observe under an inverted microscope.

[0086] 4) Add 95% ethanol at 1 mL / well and incubate for 10 min to completely dissolve the crystal violet. Pipette 100 μl of the ethanol solution containing the crystal violet into another 96-well plate. Read the A570 value with a microplate reader to quantitatively analyze the biomass of the Candida albicans biofilm.

[0087] Experimental results: Figure 7 and Figure 8 As shown, in the late stage of biofilm formation, co-incubation with 1024 μg / ml fluconazole resulted in morphological defects in C. albicans biofilms, including a decrease in the number of cells in the biofilm, reduced cross-linking between yeast and hyphae, and reduced extracellular matrix. The biofilm biomass was statistically significantly different from that in the control group (***P < 0.001). TPEN combined with fluconazole further reduced biofilm maturity, significantly reducing hyphae, and the biofilm biomass was statistically significantly different from that of either drug alone (***P < 0.001). These results indicate that TPEN enhances the inhibitory effect of fluconazole on C. albicans biofilm maturation in the late stage of biofilm formation.

[0088] Example 7 TPEN synergistically inhibits Candida albicans biofilms with fluconazole

[0089] Experimental process:

[0090] 1) Adjust the concentration of Candida albicans to 1×10 in RPMI 1640 medium. 6 CFU / mL, 100 μl of bacterial suspension was added to each well of a 96-well plate and incubated at 37°C for 2 h.

[0091] 2) Non-adherent cells were washed with saline to remove them, and 200 μl of serially diluted RPMI 1640 medium containing drugs was added to give final concentrations of fluconazole and TPEN ranging from 0.5 to 1024 μg / ml and 1.25 μM to 160 μM, respectively. Medium without drugs was used as a control.

[0092] 3) After incubation at 37°C for 24 h, the plates were gently rinsed three times with 200 μL / well PBS. 100 μL of XTT / menadione solution was added to each well and the plates were incubated at 37°C in the dark for 2 h.

[0093] 4) 80 μL of supernatant was transferred from each well to a new 96-well cell culture plate, and the A490 nm value was read using a microplate reader to qualitatively analyze the activity of the Candida albicans biofilm.

[0094] 5) Calculate the minimum inhibitory concentration (SMIC 80%), which is the lowest drug concentration in the well that reduces the colorimetric OD value by more than 80% compared to the positive control well (column 11). That is, the percentage of fungal growth inhibition = 1 - (OD value of each drug well) 490 - OD of negative control well 490 ) / (OD of positive control well 490 - OD of negative control well 490 )×100%

[0095] Experimental Results: As shown in Table 2, 80 μM TPEN inhibited biofilm activity by over 80%, resulting in a SMIC 80 of TPEN against C. albicans biofilms of 80 μM. As shown in Table 3, the SMIC 80 of fluconazole in C. albicans was greater than 1024 μg / ml. Supplementation with 5 μM TPEN reduced this to 64 μg / ml, with an FICI value of 0.125, indicating a synergistic effect (SYN) between TPEN and fluconazole. The SMIC 80 of fluconazole decreased significantly with increasing TPEN concentration, reaching <1 μg / ml at 40 μM TPEN, indicating that TPEN reversed fluconazole resistance in biofilms. These results demonstrate that TPEN can synergize with fluconazole against C. albicans biofilms and reverse azole resistance in biofilms.

[0096] Table 2 Effects of different concentrations of TPEN on anti-Candida albicans biofilm (mean ± SD, n = 3)

[0097]

[0098] aRelative absorbance = absorbance of experimental group / absorbance of control group. The ratio of the absorbance of A490 of Candida albicans biofilms treated with different concentrations of TPEN to that of the control group was determined by the XTT reduction method.

[0099] Table 3 Evaluation of the anti-Candida albicans biofilm effect of TPEN combined with fluconazole for 24 h

[0100]

[0101] a The fractional inhibitory concentration index (FICI) method is a widely used method for evaluating the interaction between combined drugs. The calculation formula is as follows: FICI = C A / MIC A +C B / MIC B , C A with C B They are the minimum inhibitory concentrations (MICs) of drugs A and B when used in combination to achieve the same efficacy. A With MIC B are the minimum inhibitory concentrations of drugs A and B when used alone, respectively.

[0102] b SYN indicates synergism; NI indicates indifference. FICI ≤ 0.5 indicates synergy, FICI > 4 indicates antagonism, and FICI between 0.5 and 4 indicates additive or indifference.

[0103] Example 8 Effect of TPEN in Fluconazole Treatment of Candida albicans Biofilm Infection

[0104] Experimental process:

[0105] 1) 48 Galleria mellonella larvae of uniform size and good vitality were selected and randomly divided into 4 groups.

[0106] 2) Use RPMI 1640 medium to adjust the concentration of Candida albicans to 1×10 6 CFU / ml, biofilms were cultured in adherent cell dishes with a diameter of 15 cm.

[0107] 3) After 24 hours, the biofilms were collected in culture dishes and centrifuged three times (3000 g, 20 min). The supernatant was removed and 5 ml of physiological saline was added to each group to prepare a biofilm suspension.

[0108] 4) Use a syringe to extract 10 μl of the biofilm suspension and inject it into the body of the greater wax moth larva through the first left anterior ventral foot. Incubate at 37°C in the dark for 2 h.

[0109] 5) Use a syringe to draw 10 μl of a solution containing 500 μg / ml fluconazole and / or 80 μM TPEN and inject it into the G. mellonella larvae through the first right anterior ventral foot. The no-drug group served as the positive control, and the group injected with only saline served as the blank control.

[0110] 6) The larvae were cultured at 37°C in the dark. Larvae that were motionless or unresponsive to slight touch were considered dead. The number of surviving larvae was recorded daily for 4 days.

[0111] Experimental results: Figure 9 As shown, after four days of hatching, the survival rates of the fluconazole and TPEN groups were significantly increased, reaching 41.7% and 33.3%, respectively, compared to the 8.33% survival rate of the WT group. The combination of TPEN and fluconazole further increased the survival rate of G. mellonella larvae to 83.33%, with statistically significant differences between the groups (P < 0.05). These results indicate that TPEN can synergize with fluconazole in the treatment of Candida albicans biofilm infections.

[0112] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions that do not depart from the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A zinc ion chelator for preparing an azole drug synergist for inhibiting Candida albicans biofilm, characterized in that: The zinc ion chelator is N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, and the azole drug is selected from fluconazole. The concentration of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine is 2.5 uM, the concentration of fluconazole is 256 ug / mL, or, The concentration of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine is 5 uM, the concentration of fluconazole is 64 ug / mL, or, The concentration of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine is 10 uM, the concentration of fluconazole is 64 ug / mL, or, The concentration of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine was 20 uM, and the concentration of fluconazole was 4 ug / mL.

2. A composition for inhibiting Candida albicans biofilm, characterized in that: The efficacy components of the composition are N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine and fluconazole. The concentration of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine is 2.5 uM, the concentration of fluconazole is 256 ug / mL, or, The concentration of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine is 5 uM, the concentration of fluconazole is 64 ug / mL, or, The concentration of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine is 10 uM, the concentration of fluconazole is 64 ug / mL, or, The concentration of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine was 20 uM, and the concentration of fluconazole was 4 ug / mL.

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