Application of PBT2 in preparation of anti-candida albicans biological membrane medicine

Abstract

The invention discloses an application of PBT2 in preparation of an anti-candida albicans biofilm drug. The invention provides the whole process that the PBT2 can inhibit the formation of a candida albicans biofilm, and the PBT2 can be combined with fluconazole to play a synergistic effect. The PBT2 reduces the contents of Zn < 2 + >, Cu < 2 + > and Fe < 2 + > in the candida albicans, seriously interferes the steady state of metal ions in candida albicans cells, further affects the normal physiological functions of the candida albicans and inhibits the formation of a candida albicans biological membrane. According to the invention, the in-vivo antibacterial effect of the PBT2 is preliminarily verified in a greater wax moth larva model. The invention provides a new strategy for eliminating Candida albicans biofilm infection, and based on the confirmed safety of PBT2, the transformation of antifungal application of PBT2 can be quickly promoted.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of PBT2 in the preparation of anti-Candida albicans biofilm drugs. Background Art

[0002] Candida albicans is a fungus that is widely found in nature and in the human body. Infections with Candida albicans are often associated with a weakened immune system, and up to 80% of infections are related to the biofilms it forms.

[0003] Candida albicans is one of the most common opportunistic pathogenic fungi and can parasitize on the skin, mouth, intestines, vagina and other parts of the body as part of the normal human microbiome. In 2022, WHO released its first list of priority fungal pathogens. Candida albicans was listed in the "critical priority group" due to its high public health burden and called for increased investment in research on Candida albicans. In recent years, with the extensive use of immunosuppressants, broad-spectrum antimicrobial drugs, various biological agents, and the development of various invasive procedures in the surgical field, Candida albicans has become more likely to transform from a harmless colonizer into a pathogen. Candida albicans is already the third most commonly isolated microorganism in blood infections in hospitalized patients, and even with effective antifungal treatment, its mortality rate is still as high as 20-50%.

[0004] Although drug-resistant isolates of Candida albicans are relatively uncommon in clinical practice, the frequent formation of biofilms on the surfaces of medical devices exponentially increases the difficulty of treatment. This is because biofilm formation increases drug resistance by secreting extracellular matrix to form a physical barrier, upregulating multidrug-resistance genes, and altering cellular metabolism. Studies have shown that Candida isolated from biofilms exhibit significantly increased resistance to common antifungal drugs such as azoles and echinocandins. Therefore, there is an urgent need to develop new antifungal drugs to enrich clinicians' arsenals.

[0005] 8-Hydroxyquinoline analog PBT2 (5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline) is an orally safe copper / zinc ion carrier with the chemical formula Initially developed for the treatment of neurodegenerative diseases, PBT2 modifies the bioavailability and distribution of metal ions such as copper and zinc to treat neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. It has demonstrated good safety and tolerability at a dose of 250 mg / day. In recent years, researchers have discovered that PBT2 has a broad antimicrobial spectrum, including against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococci, and Klebsiella pneumoniae. Recent studies have demonstrated that PBT2 can act as an ion carrier, mediating the excessive accumulation of zinc ions in bacteria, thereby disrupting the homeostasis of metal ions such as manganese and iron, thereby exerting its antimicrobial effects. However, there have been no reports of PBT2 being used to eliminate fungi such as Candida albicans. Summary of the Invention

[0006] The object of the present invention is to overcome at least one deficiency of the prior art and provide an application of PBT2 in the preparation of an anti-Candida albicans biofilm drug.

[0007] The technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides a use of an 8-hydroxyquinoline analogue in the preparation of a drug for resisting Candida albicans biofilm.

[0009] In some examples, the 8-hydroxyquinoline analog is selected from PBT2.

[0010] In a second aspect, the present invention provides a use of an 8-hydroxyquinoline analogue in the preparation of a synergist for azole drugs against Candida albicans biofilms.

[0011] In some examples, the 8-hydroxyquinoline analog is selected from PBT2.

[0012] In some examples, the azole drug is selected from fluconazole.

[0013] In some embodiments, the 8-hydroxyquinoline analog further comprises a pharmaceutically acceptable solvate of PBT2.

[0014] In some examples, the drug is administered topically, orally, by injection, implant, rectum, spray, or inhalation.

[0015] 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.

[0016] In some examples, the drug further includes a pharmaceutical carrier or a pharmaceutically acceptable excipient.

[0017] The beneficial effects of the present invention are:

[0018] 1) PBT2 has inhibitory effects on both Candida albicans planktonic bacteria and Candida albicans biofilms, which broadens the antibacterial spectrum and usage scenarios of compound PBT2.

[0019] 2) PBT2 combined with the commonly used antifungal drug fluconazole can more effectively eliminate Candida albicans biofilm.

[0020] 3) The present invention proposes that PBT2 affects the metal ion homeostasis of Candida albicans and reduces the Cu 2+ 、Fe 2+ ,、Zn 2+ The content of β-actin affects the normal physiological function of Candida albicans and the formation of biofilm, providing a new idea for the removal of drug-resistant biofilm.

[0021] 4) PBT2 and fluconazole have different mechanisms of action, and combined use of different targets can reduce the possibility of Candida albicans developing drug resistance.

[0022] 5) Reducing 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 and provide a safer biofilm treatment solution.

[0023] 6) Prospects for clinical application: The safety of PBT2 has been previously verified in Phase II clinical trials, and previous studies have shown its inhibitory effects on a variety of bacteria. This study further expands its scope of use to Candida albicans, further expanding its anti-infection application range from bacteria to fungi, and is expected to be promoted clinically in a relatively short period of time. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the time inhibition curve of PBT2 at different concentrations on Candida albicans planktonic bacteria in Example 1.

[0025] Figure 2 Different concentrations of PBT2 (a: 128 μM PBT2, b: 64 μM PBT2, c: 32 μM PBT2, d: 16 μM PBT2, e: control group) inhibited the early adhesion process of Candida albicans biofilm formation.

[0026] Figure 3 Different concentrations of PBT2 (a: 128 μM PBT2, b: 64 μM PBT2, c: 32 μM PBT2, d: 16 μM PBT2, e: control group) inhibited the yeast-hyphae transformation in the middle stage of Candida albicans biofilm formation.

[0027] Figure 4Different concentrations of PBT2 (a: 128 μM PBT2, b: 64 μM PBT2, c: 32 μM PBT2, d: 16 μM PBT2, e: control group) inhibit the maturation process of the three-dimensional structure of the Candida albicans biofilm in the late stage.

[0028] Figure 5 The combined inhibitory effect of PBT2 and fluconazole (FLC) on Candida albicans biofilm.

[0029] Figure 6 For metal ions (Cu 2+ ,Fe 3+ ,Zn 2+ ) rescue effect of PBT2 against Candida albicans.

[0030] Figure 7 This is the effect diagram of PBT2 reducing the content of zinc ions in Candida albicans cells.

[0031] Figure 8 This is the effect diagram of PBT2 reducing the ferrous ion content in Candida albicans.

[0032] Figure 9 This is the effect diagram of PBT2 reducing the copper ion content in Candida albicans.

[0033] Figure 10 This figure shows the effect of PBT2 on reducing the in vivo virulence of Candida albicans in a simulated infection model of the greater wax moth larvae. DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited thereto.

[0035] The experimental materials and reagents used in the following examples of the present invention are shown in Table 1, and the sources of experimental instruments are shown in Table 2.

[0036] Table 1

[0037]

[0038]

[0039] Table 2

[0040]

[0041] Example 1

[0042] Experimental process:

[0043] 1) 1.0×10 4 cell·mL -1The Candida albicans was resuspended in 5 mL of YPD liquid medium containing different concentrations of PBT2 (128 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM) and cultured at 37 °C with continuous shaking (180 rpm). YPD liquid medium without drug was used as a positive control;

[0044] 2) After thoroughly mixing the samples at fixed time points (0, 4, 8, 16, and 24 h), 100 μL of bacterial suspension was taken from each group and diluted 10-fold in normal saline;

[0045] 3) Spread the diluted bacterial suspension on YPD agar plates and place the plates in a 37°C incubator;

[0046] 4) Count the colony forming units (CFU) after 36 hours.

[0047] Experimental results: Figure 1 As shown, in YPD medium, 4 μM PBT2 did not exhibit obvious anti-Candida albicans effect, and PBT2 showed concentration-dependent inhibition on Candida albicans at concentrations of 8-128 μM, but did not exhibit bactericidal effect as the concentration increased.

[0048] Example 2

[0049] Experimental process:

[0050] 1) 1×10 6 CFU / mL Candida albicans was resuspended in 1 mL of RPMI 1640 medium containing different concentrations of PBT2, and medium without drug was used as control, and added to 24-well plates and incubated for 2 h.

[0051] 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.

[0052] 3) Add 95% ethanol at a rate of 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.

[0053] Experimental results: Figure 2 As shown in the figure, in the early stage of biofilm formation, 16μM-128μM PBT2 significantly inhibited the adhesion of Candida albicans, and the difference was statistically significant compared with the control group (***P<0.001). The above results indicate that PBT2 has an inhibitory effect on the adhesion of Candida albicans in the early stage of biofilm formation.

[0054] Example 3

[0055] Experimental process:

[0056] 1) Prepare RPMI 1640 culture medium (containing 10% fetal bovine serum) containing different concentrations of PBT2. Culture medium without drug was used as control. A final concentration of 1×10 6 CFU / mL of Candida albicans was cultured at 37 °C with shaking at 180 rpm for 4 h to induce hyphae formation of Candida albicans.

[0057] 2) Take 1 mL of bacterial suspension from each group and add it to a 24-well plate, and observe under an inverted microscope.

[0058] Experimental results: Figure 3 As shown, 16 μM-128 μM PBT2 inhibited the budding, hyphae formation and elongation of Candida albicans in a concentration-dependent manner, and inhibited the yeast-hyphae transition process in the middle stage of Candida albicans biofilm.

[0059] Example 4

[0060] Experimental process:

[0061] 1) Adjust the concentration of Candida albicans to 1×10 in RPMI 1640 medium. 6 CFU / mL, 1 ml of bacterial suspension was added to each well of a 24-well plate and incubated at 37 °C for 2 h to construct a biofilm model.

[0062] 2) Wash the non-adherent cells with physiological saline, and add 1 mL of RPMI 1640 medium containing different concentrations of PBT2 to each well. Medium without drug was used as a control.

[0063] 3) After incubation at 37°C for 24 h, non-adherent cells were removed with physiological saline, fixed with 1 mL / well methanol, stained with 0.1% crystal violet, rinsed with double-distilled water, dried at room temperature, and observed under an inverted microscope.

[0064] 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.

[0065] Experimental results: Figure 4 As shown in the data, with the increase of PBT2 concentration, the density and layer of biofilm cells gradually decreased, and the cell morphology was mostly spore and pseudohyphae. Compared with the control group, mature biofilm could not be formed. The biomass of biofilm was significantly different from that of the control group (**P < 0.01, ***P < 0.001), indicating that PBT2 has an inhibitory effect on the late three-dimensional structure maturation of Candida albicans biofilm.

[0066] Example 5

[0067] Experimental process:

[0068] 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.

[0069] 2) Non-adherent cells were removed by washing with saline, and 200 μL of drug-containing RPMI 1640 medium was added to adjust the final concentrations of fluconazole and PBT2 to 64 to 1024 μg / ml and 64 μM to 256 μM, respectively. A 64 μg / ml fluconazole + 64 μM PBT2 combination treatment group was established, and medium without drug was used as a control.

[0070] 3) After incubation at 37°C for 24 h, the cells were gently rinsed three times with 200 μL / well PBS. 200 μL of XTT / menadione solution was added to each well and the cells were incubated at 37°C in the dark for 2 h.

[0071] 4) 180 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.

[0072] Experimental results: Figure 5 As shown in the results, 64 μg / ml fluconazole could reduce the metabolic activity of Candida albicans biofilm by 64.0%±5.21%. However, the anti-biofilm effect did not change significantly with the increase of fluconazole concentration. The biofilm activity of Candida albicans was reduced by 61.9%±1.10% when 64 μg / ml fluconazole was used in combination with 64 μM PBT2 compared with the activity of 64 μg / ml fluconazole alone, indicating that PBT2 can assist fluconazole in further reducing the biofilm activity of Candida albicans.

[0073] Example 6

[0074] Experimental process:

[0075] RPMI 1640 medium containing PBT2 was serially diluted in a 96-well plate to a concentration range of 4 μM to 64 μM in a volume of 100 μL. Medium without drug was used as a control.

[0076] Prepare 4-fold working concentrations of copper, iron, and zinc solutions in RPMI 1640 medium and add 50 μL / well to a 96-well plate;

[0077] The concentration of Candida albicans in RPMI1640 medium was adjusted to 4×10 3 cell·mL -1 , add 50 μL / well to a 96-well plate;

[0078] The 96-well plate was placed in a 37°C constant temperature incubator in the dark for 36 h and observed under an inverted microscope.

[0079] Experimental results: Figure 6 As shown in the data, when no metal ions were added, PBT2 concentrations of 4 μM and above completely inhibited colony formation, and only a small amount of hyphae were visible; after the addition of zinc ions, Candida albicans was still able to form mature colonies when co-incubated with 8 μM PBT2, and still formed smaller colonies when co-incubated with 16 μM PBT2; similarly, the addition of copper ions or iron ions could also reduce the antibacterial effect of PBT2 to a certain extent, and the mixed addition of the above metal ions could rescue the inhibitory effect of PBT2 on colony formation to the greatest extent.

[0080] Example 7

[0081] Experimental process:

[0082] 1) Dissolve zinc ion fluorescent probe Zinquin ethyl ester powder in DMSO solution and adjust the concentration to 10 mM to prepare the Zinquin ethyl ester stock solution.

[0083] 2) Add zinc ion solution to RPMI 1640 medium to adjust the final zinc ion concentration to 200 μM.

[0084] 3) Add 1×10 7 albicans cells / ml, and RPMI 1640 medium containing zinc ions without drugs was used as a control.

[0085] 4) Incubate at 37°C with shaking at 180 rpm for 16 h. Centrifuge the bacterial suspension for 10 min (3000 rpm), discard the supernatant, resuspend in saline, and centrifuge again. Repeat this process three times.

[0086] 5) Before use, dilute the Zinquin ethyl ester stock solution with DMSO to a 40 μM working solution. Resuspend the cells in the Zinquin ethyl ester 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.

[0087] 6) 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.

[0088] Experimental results: Figure 7As shown in the figure, with the increase of PBT2 concentration, the fluorescence intensity of zinc ions in Candida albicans cells decreased in a concentration-dependent manner, and the difference was statistically significant compared with the control group (***P<0.001), indicating that PBT2 reduced the zinc ion level in Candida albicans cells.

[0089] Example 8

[0090] Experimental process:

[0091] 1) Place the ferrous ion fluorescent probe FeRhoNox-1 powder at room temperature for 30 minutes and then dissolve it in DMSO solution. The concentration is adjusted to 1 mM to prepare the FeRhoNox-1 storage solution.

[0092] 2) Add iron ion solution to RPMI 1640 medium to adjust the final zinc ion concentration to 400 μM.

[0093] 3) Add 1×10 7 albicans cells / ml, and RPMI 1640 medium containing iron ions without drugs was used as a control.

[0094] 4) Incubate at 37°C with shaking at 180 rpm for 16 h. Centrifuge the bacterial suspension for 10 min (3000 rpm), discard the supernatant, resuspend in saline, and centrifuge again. Repeat this process three times.

[0095] 5) Before use, dilute the FeRhoNox-1 stock solution with DMSO to a 5 μM FeRhoNox-1 working solution. Resuspend the cells in FeRhoNox-1 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 saline, vortex to mix, and centrifuge again. Repeat this process three times to remove any residual staining solution.

[0096] 6) 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 532 nm and an emission wavelength of 570 nm to read the fluorescence intensity of each experimental group.

[0097] Experimental results: Figure 8 As shown in the figure, the fluorescence intensity of intracellular ferrous ions in Candida albicans was slightly reduced after incubation with 64 μM PBT2, and the difference was statistically significant compared with the control group (**P < 0.01), while the fluorescence intensity did not change significantly when incubated with 32 μM PBT2 and 16 μM PBT2, indicating that higher concentrations of PBT2 can reduce the level of ferrous ions in Candida albicans cells to a certain extent.

[0098] Example 9

[0099] Experimental process:

[0100] 1) Add copper ion solution to RPMI 1640 medium to adjust the final zinc ion concentration to 10 μM.

[0101] 2) Add 1×10 8 albicans cells / ml, and RPMI 1640 medium containing copper ions without drugs was used as a control.

[0102] 2) Incubate at 37°C with shaking at 180 rpm for 16 h. Centrifuge the bacterial suspension for 10 min (3000 rpm, 4°C), discard the supernatant, resuspend in saline, and centrifuge again. Repeat the centrifugation three times.

[0103] 3) After removing the supernatant, resuspend the Candida albicans in double-distilled water and add it to a grinding tube. After adding zirconium beads, the tube was placed in a grinder and ground into a homogenate (70 Hz, 60 s on, 30 s off, repeated 5 times).

[0104] 4) Centrifuge the homogenate for 5 min (6000 rpm, 4°C) and take the supernatant for testing.

[0105] 5) According to copper (Cu 2+ ) The instructions for the colorimetric test kit instructed to determine the copper ion concentration of the supernatant, and at the same time, the BCA protein quantification method was used to determine the protein content of the supernatant as a standard.

[0106] Experimental results: Figure 9 As shown in the results, the intracellular copper ion content of Candida albicans was significantly reduced after incubation with 64μM and 32μM PBT2, and the difference was statistically significant compared with the control group (**P<0.01). However, the copper ion content did not change significantly when incubated with 16μM PBT2, indicating that PBT2 concentrations above 32μM can significantly reduce the copper ion level in Candida albicans cells.

[0107] Example 10

[0108] Experimental process:

[0109] 1) 100 Galleria mellonella larvae of uniform size and good vitality were selected and randomly divided into 5 groups.

[0110] 2) Use RPMI 1640 medium to adjust the concentration of Candida albicans to 2×10 7 cell·mL -1 , draw 10 μL of Candida albicans solution with a syringe, inject it into the body of the greater wax moth larvae through the left front first abdominal foot, and incubate at 37°C for 2 h.

[0111] 5) Use a syringe to draw 10 μL of solution containing different drugs and inject it into the body of the greater wax moth larvae through the first right anterior abdominal foot. The drug-free group serves as the positive control group.

[0112] 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.

[0113] Experimental results: Figure 10 As shown, after 4 days of observation, the survival rate of the greater wax moth in the control group was only 15%, while that of the PBT2 treatment groups (360 ng / larva, 180 ng / larva, 90 ng / larva) was 85%, 60%, and 35%, respectively. In comparison, the survival rate of the fluconazole treatment group (4 μg / larva) was 65%, which preliminarily verified the effect of PBT2 in treating Candida albicans infection in vivo.

[0114] 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. Use of an 8-hydroxyquinoline analogue in the preparation of a drug for resisting Candida albicans biofilm.

2. The use according to claim 1, characterized in that The 8-hydroxyquinoline analogue is selected from PBT2.

3. Application of an 8-hydroxyquinoline analogue in the preparation of an azole drug synergist for anti-Candida albicans biofilm.

4. The use according to claim 3, characterized in that The 8-hydroxyquinoline analogue is selected from PBT2.

5. The use according to claim 3, characterized in that The azole drug is selected from fluconazole.

6. The use according to any one of claims 1 or 3, characterized in that The 8-hydroxyquinoline analogs also include pharmaceutically acceptable solvates of PBT2.

7. The use according to any one of claims 1 or 3, characterized in that The drug is an external administration agent, an oral administration agent, an injection administration agent, an implantation administration agent, a rectal administration agent, a spray administration agent or an inhalation administration agent.

8. The use according to any one of claims 1 or 3, characterized in that 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.

9. The use according to any one of claims 1 or 3, characterized in that The drug further includes a drug carrier or a pharmaceutically acceptable excipient.

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