Use of carbonyl cyanide-4-trifluoromethoxyphenylhydrazone in the prevention and treatment of candida infections
Carbonyl cyanide-4-trifluoromethoxyphenylhydrazone addresses the problem of antifungal drug resistance in Candida infections by disrupting the mitochondria of Candida and inhibiting biofilms, thus providing an effective treatment option.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-13
- Publication Date
- 2026-07-28
AI Technical Summary
Candida albicans and Candida auris are highly resistant to existing antifungal drugs, making treatment difficult. In particular, Candida auris is resistant to multiple drugs, and the formation of biofilms leads to drug penetration and excretion, limiting the effectiveness of traditional therapies.
Carbonyl cyanide-4-trifluoromethoxyphenylhydrazone (FCCP) is used as the active ingredient to prepare drugs for the prevention or treatment of Candida infections. It enhances the antibacterial effect by disrupting the mitochondria of Candida and inhibiting biofilm growth.
FCCP showed significant antibacterial activity against multidrug-resistant Candida strains, effectively inhibiting colony growth and biofilm formation, and disrupting mitochondria, providing a new treatment option for Candida infections and avoiding antibiotic resistance.
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Abstract
Description
Technical Field
[0001] This application belongs to the field of Candida infection prevention and control technology, and specifically relates to the use of 4-trifluoromethoxyphenylhydrazone in the prevention and control of Candida infection. Background Technology
[0002] Candida albicans and Candida auris belong to the genus Candida. Candida albicans is an opportunistic pathogenic fungus, scientifically known as... Candida albicans It is a common member of the human microbiome, usually existing in a harmless symbiotic form in the mouth, gastrointestinal tract, vagina, and skin surface of healthy individuals. Candida albicans can switch between a yeast phase and a hyphal phase depending on environmental conditions. The yeast phase is an oval, single-celled morphology, typically associated with symbiosis and harmless reproduction. The hyphal phase usually forms long, branched hyphae that can invade human tissues and is closely related to pathogenicity. This morphological conversion is key to its ability to overcome physiological barriers and cause infection. Candida auris, scientifically known as *Candida auris*, is a relatively recently discovered member of the Candida family. Since its initial discovery in the ear canal secretions of a patient in Japan in 2009, it has rapidly become a key emerging pathogen of global public health concern. Its core threat lies not in ordinary infection, but in its unique biological characteristics, which pose a serious risk to critically ill patients in medical settings such as hospitals. Candida auris exhibits significant multidrug resistance, with many strains naturally resistant to commonly used first-line antifungal drugs (such as fluconazole). Some strains even show resistance to all three major classes of antifungal drugs (azoles, echinocandins, and polyenes), which greatly limits treatment options and can lead to treatment failure. Therefore, it is often referred to as a "super fungus" in the media.
[0003] Candidiasis occurs when Candida albicans overgrows and invades tissues. Infections can range from superficial, localized mucosal infections to life-threatening systemic infections. Superficial / mucosal candidiasis is the most common type, affecting both the skin and mucous membranes. Examples include oral candidiasis (commonly known as thrush), which is common in infants, denture wearers, those using inhaled corticosteroids, or those with immunosuppression. Symptoms include white patches on the oral mucosa and tongue, which may reveal red lesions after scraping. Vulvovaginal candidiasis (commonly known as yeast infection) is something many women experience at least once in their lifetime. Symptoms include vulvar itching, burning, white, cottage cheese-like discharge, and redness. Candidal balanitis is a male genital infection characterized by erythema, itching, and discharge on the glans penis. Cutaneous candidiasis often occurs in warm, moist skin folds (such as the armpits, groin, between the fingers, and under the breasts), presenting as red macules or papules accompanied by itching or burning. Paronychia and onychomycosis: These affect the nail and surrounding tissues, causing redness, swelling, and pain around the nail. The nail may also thicken and discolor.
[0004] Invasive candidiasis is a serious systemic infection in which the fungus enters the bloodstream and spreads to organs throughout the body (such as the heart, brain, eyes, kidneys, and bones), with a high mortality rate. Examples include candidemia, where Candida is present in the blood, and is the most common invasive form. It is often caused by medical devices such as central venous catheters. Deep organ infections include hepatobiliary candidiasis, candidal endocarditis, candidal meningitis, and endophthalmitis.
[0005] Candida auris can colonize human skin and mucous membranes for extended periods and is resistant to many common disinfectants. This allows it to spread and cause outbreaks within healthcare facilities (especially in intensive care units) through contact with contaminated environments or objects. Candida auris can infect local areas of the body, commonly found in the ear canal and wounds, causing ear pain, purulent discharge, itching, and hearing loss. However, the most dangerous situation is when it enters the bloodstream, causing candidemia, or spreads to internal organs. Symptoms include persistent high fever, chills, and organ failure. Invasive infections have a high mortality rate.
[0006] Both *Candida albicans* and *Candida auris* can form highly drug-resistant biofilms, which is the core mechanism by which they cause intractable infections. Biofilms consist of fungal cells and their secreted extracellular matrix (EPS), with β-1,3-glucan as the main component. This effectively captures and prevents antifungal drugs (such as fluconazole and amphotericin B) from penetrating deeper cells. Furthermore, metabolically inert cells exist within the biofilm, exhibiting natural resistance to drugs targeting active proliferation processes. Simultaneously, drug efflux pumps such as the ATP-binding cassette (ABC) and the major facilitater superfamily (MFS) are significantly upregulated in the biofilm state, actively expelling drugs extracellularly. *Candida auris* is particularly problematic; most clinical strains exhibit inherent resistance to azole drugs and readily develop acquired resistance to echinocandins. Its biofilm structure is denser, forms faster, and can survive for extended periods on medical device surfaces, greatly facilitating nosocomial transmission. In contrast, *Candida albicans* relies on yeast-hyphae transition to construct its biofilm framework; while exhibiting strong resistance, it is generally less prevalent than that of *Candida auris*. The shared drug resistance mechanism of both highlights the limitations of traditional antifungal therapies.
[0007] Currently, the treatment of both Candida albicans and Candida auris infections stems from their drug resistance. This is mainly due to their resistance to existing drugs and the limitations of the drugs themselves. Clinically, three main classes of antifungal drugs are currently relied upon: azoles (such as fluconazole, but Candida auris has a very high resistance rate to them), echinocandins (such as caspofungin, which is currently the first-line drug of choice for invasive infections, but resistance may still occur), and polyenes (such as amphotericin B, but it has significant side effects such as nephrotoxicity). Summary of the Invention
[0008] To address the aforementioned issues, this application provides the use of 4-trifluoromethoxyphenylhydrazone in the prevention and treatment of Candida infections.
[0009] The first object of this application is to provide the use of 4-trifluoromethoxyphenylhydrazone or a pharmaceutically acceptable salt thereof as the sole active ingredient in any of the following: (i) A medicine for the preparation of a preventive or therapeutic drug for diseases caused by Candida infection, wherein the Candida is Candida albicans and / or Candida auris; (ii) Used to prepare antibacterial and / or bactericidal products for Candida, wherein the Candida is Candida albicans and / or Candida auris.
[0010] Furthermore, the Candida species is Candida albicans SC5314.
[0011] Furthermore, the minimum inhibitory concentration of the 4-trifluoromethoxyphenylhydrazone against wild-type Candida albicans SC5314 is 32 μg / mL.
[0012] Furthermore, the Candida species is a drug-resistant bacterium resistant to antifungal drugs.
[0013] Further, the antifungal drug is selected from azole antifungal drugs, echinocandins antifungal drugs, and polyene antifungal drugs; preferably, it is an azole antifungal drug, wherein azoles include imidazoles and triazoles, imidazoles including but not limited to ketoconazole, miconazole, econazole, and clotrimazole; triazoles including but not limited to itraconazole, voriconazole, fluconazole, posaconazole, and lavconazole. More preferably, the antibiotic is a triazole, such as itraconazole and voriconazole.
[0014] Furthermore, the Candida species is an azole-resistant Candida albicans.
[0015] Furthermore, the minimum inhibitory concentration of the 4-trifluoromethoxyphenylhydrazone against azole-resistant Candida albicans is 32 μg / mL.
[0016] Furthermore, the Candida species is azole-resistant Candida auris.
[0017] Furthermore, the minimum inhibitory concentration of the 4-trifluoromethoxyphenylhydrazone against azole-resistant Candida auris is 16 μg / mL.
[0018] Furthermore, the drug is a drug that inhibits the growth of Candida.
[0019] Furthermore, the drug is a drug that inhibits the growth of Candida biofilm.
[0020] Furthermore, the drug is a drug that destroys the mitochondria of Candida.
[0021] To achieve the intended therapeutic purpose and enhance the antibacterial effect, the drug may be administered using any known method of administration, including but not limited to oral administration, injection, sublingual administration, or oral spray administration. Depending on the desired antibacterial effect, the nature and severity of the disease to be treated, and the individual circumstances of the patient or animal, the method of administration and dosage may vary considerably; for example, the dosage may be set in the range of 5–25 μM.
[0022] Compared with the prior art, this application has the following advantages: The application of 4-trifluoromethoxyphenylhydrazone (FCCP) in the prevention and treatment of Candida infections was investigated for the first time. The antibacterial activity, anti-biofilm effect, and mitochondrial disruption of FCCP against Candida albicans and Candida auris (including multidrug-resistant strains) were tested. In the in vitro antibacterial experiment, the antibacterial effect of FCCP against Candida achieved unexpected technical results. Its minimum inhibitory concentration and other related in vitro indicators were significantly higher than those of the classic antifungal drug fluconazole.
[0023] Furthermore, by staining with the mitochondrial dye Mito-Tracker and combining it with Nikon turntable fluorescence confocal microscopy, we were able to visually confirm that FCCP can specifically and efficiently destroy the mitochondrial morphology of Candida auris, leading to a decrease in its growth ability, highlighting its selective targeting ability to fungal mitochondria.
[0024] The above experimental results demonstrate that FCCP exhibits good inhibition of cannula growth, good inhibition of biofilm (colony growth), and good mitochondrial disruption (conidia growth) in antibacterial experiments against various Candida species (wild-type Candida albicans, drug-resistant Candida albicans, and drug-resistant Candida auris). This provides a new therapeutic agent for Candida albicans and Candida auris infections, while simultaneously addressing the clinical problem of antibiotic resistance in these infections, thus avoiding the drawback of antibiotics easily developing resistance.
[0025] Other features and advantages of this application will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the application. The objectives and other advantages of this application may be realized and obtained by means of the structures pointed out in the description, claims and drawings. Attached Figure Description
[0026] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0027] Figure 1 The diagram shows the FCCP MIC detection pattern according to Embodiment 1 of this application; Figure 2 The following diagram shows the flu MIC detection pattern according to Embodiment 1 of this application; Figure 3 The figure shows the experimental results of the effect of FCCP on Candida growth according to Example 2 of this application. Figure 4 The following figure shows the experimental results of the effect of flu on the growth of Candida according to Example 2 of this application; Figure 5 The graph shows the CFW test results of strains SC5314 and 5172 according to Example 3 of this application; Figure 6 The graph shows the CFW test results of strains C1, C2 and 12373 according to Example 3 of this application; Figure 7 The diagram shows the mitochondrial dye test results of strains SC5314 and 5172 according to Example 4 of this application; Figure 8 The diagram shows the mitochondrial dye test results of strains C1, C2 and 12373 according to Example 4 of this application. Detailed Implementation
[0028] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0029] Experimental materials 1. Strains: The Candida albicans used in this experiment was wild-type Candida albicans SC5314; and isolate 5172, which is resistant to clinical azole drugs (itraconazole and voriconazole).
[0030] The Candida auris strains used in this experiment were isolates 12373, C1, and C2 that were resistant to clinical azole drugs (itraconazole and voriconazole).
[0031] 2. The composition of the culture medium and mitochondrial dyes is detailed in Table 1. Table 1
[0032] Example 1 Determination of the minimum inhibitory concentration (MIC) of FCCP and flu against each strain: Test method: Adjust the concentration of each bacterial culture to 2×10⁻⁶ using RPMI-1640 liquid medium. 3 Cells / mL, each drug solution was serially diluted 100 μL and added sequentially to a 96-well plate, followed by 100 μL of bacterial suspension in each well; the plates were then incubated at 37 ℃ for 48 h, and the minimum inhibitory concentration (MIC) of the drug against Candida was observed visually. The experiment was repeated three times. The test results are as follows. Figure 1 and Figure 2 As shown.
[0033] Figure 1 For FCCP MIC detection, Figure 2 The MIC of flu (abbreviation for fluconazole, used as a control group) was measured. Figure 1 and Figure 2 From top to bottom, they are different strains. Figure 1 In the middle, the FCCP groups are arranged from left to right as different FCCP concentrations (0, 4, 8, 16, 32, 42, 64, 128 ug / mL). Figure 2 In the middle, the flu group, from left to right, represents different flu concentrations (0, 4, 8, 16, 32, 64, 128, 256, 512, 1024 ug / mL).
[0034] from Figure 1 and Figure 2 The results show that FCCP has a MIC of 32 ug / mL against Candida albicans SC5314 and 5172, and a MIC of 16 ug / mL against Candida auris C1, C2, and 12373. flu has a MIC of 1024 ug / mL against Candida auris C1 and C2, 128 ug / mL against 5172, 128 ug / mL against 12373, and 2 ug / mL against Candida albicans SC5314. This demonstrates that FCCP has a stronger inhibitory effect on Candida albicans C1, C2, 12373, and 5172 than flu.
[0035] Example 2 Experiment on the effects of FCCP and flu on Candida growth Experimental method: The concentration of each bacterial culture was adjusted to 10 using a gradient of YPD liquid culture medium. 8 10 7 10 6 10 5 10 4 10 3cells / mL. Different concentrations and species of bacterial suspensions were added dropwise to YPD solid medium containing different concentrations of different drugs in sequence and incubated at 30℃ for 48 h, followed by observation of growth. The experiment was repeated three times. Specifically, when using flu, the concentrations for strains SC5314 and 5172 were 0, 20, and 40 μM, respectively; and the concentrations for strains C1, C2, and 12373 were 0 μM, 10 μM, and 20 μM, respectively. When using FCCP, the concentrations for strains SC5314 and 5172 were 0, 20, and 40 μM, respectively; and the concentrations for strains C1, C2, and 12373 were 0 μM, 10 μM, and 20 μM, respectively.
[0036] Experimental results are as follows Figure 3 and Figure 4 As shown. Figure 3 This is a graph showing the experimental results of the effect of FCCP on Candida growth. Figure 3 In the diagram, at each drug concentration, from left to right, the values are 10. 3 10 4 10 5 10 6 10 7 10 8 Bacterial solution with a concentration of cells / mL. Figure 4 This is a diagram showing the experimental results of the effect of flu on the growth of Candida. Figure 4 In the diagram, at each drug concentration, from left to right, the values are 10. 3 10 4 10 5 10 6 10 7 10 8 cells / mL.
[0037] from Figure 3 It can be seen that the growth of C1, C2, and 12373 cells was not significantly inhibited by Flu at 20 μM and 10 μM compared to the control group. In the control group, the concentration of C1 and C2 bacterial cultures reached 10 μM. 3 Growth can be observed at a concentration of cells / mL, and the concentration of 12373 bacterial culture reaches 10. 5 Growth could be observed at cell / mL, and the bacterial concentrations of C1 and C2 in the 20 μM and 10 μM groups reached 10. 3 Growth can be observed at a concentration of cells / mL, and the concentration of 12373 bacterial culture reaches 10. 5 Growth can be observed when the cell / mL ratio is reached.
[0038] from Figure 4 It can be seen that the growth of C1, C2, and 12373 cells in 20 μM and 10 μM FCCP was significantly inhibited compared with the control group. In the control group, the concentration of C1 and C2 bacterial suspensions reached 10 μM.3 Growth can be observed when the bacterial concentration of 12373 reaches 10 cells / mL; 5 Growth could be observed at cell / mL; at a concentration of 20 uM FCCP, C1 and C2 showed growth when the bacterial concentration reached 10. 7 At a bacterial concentration of 10 cells / mL, growth was weak; 12373 showed weak growth even when the bacterial concentration reached 10. 8 When the number of cells / mL is low, growth is weak.
[0039] from Figure 3 The results showed that the growth of SC5314 was significantly inhibited by Flu at 40 μM and 20 μM compared to the control group, while no significant inhibition was observed in the drug-resistant bacterium 5172. In the control group, the SC5314 bacterial concentration reached 10 μM. 3 Growth could be observed at a concentration of 10 cells / mL, and the concentration of 5172 bacteria reached 10. 4 Growth could be observed at cell / mL; the SC5314 bacterial concentration reached 10 in the 40 uM and 20 uM groups. 7 Growth could be observed at a concentration of 10 cells / mL, and the concentration of 5172 bacteria reached 10. 4 Growth can be observed when the cell / mL ratio is reached.
[0040] from Figure 4 The results showed that the growth of SC5314 was significantly inhibited by FCCP at 40 μM and 20 μM compared to the control group, while no significant inhibition was observed in the drug-resistant bacterium 5172. In the control group, the SC5314 bacterial concentration reached 10... 3 Growth could be observed at a concentration of 10 cells / mL, and the concentration of 5172 bacteria reached 10. 4 Growth could be observed at cell / mL; the SC5314 bacterial concentration reached 10 in the 40 uM and 20 uM groups. 6 Growth could be observed at a concentration of 10 cells / mL, and the concentration of 5172 bacteria reached 10. 4 Growth can be observed when the cell / mL ratio is reached.
[0041] from Figure 3 and Figure 4 The comparison shows that, at the same concentration, FCCP inhibits the growth of SC5314 and 5172 similarly to Flu; at the same concentration, FCCP inhibits the growth of C1, C2, and 12373 more strongly than Flu.
[0042] Example 3 The effect of FCCP on the biofilm content of Candida auris was determined using the calcium fluorescent white (CFW) method. Test method: Adjust the concentration of each bacterial culture to 2.0 × 10⁻⁶ using RPMI-1640 medium. 3 cells / mL. Control group (no drug added), 10 μM drug-treated group, and 20 μM drug-treated group, using either FCCP or flu. 2 mL of each bacterial culture was placed in a confocal microplate and incubated at 37°C for 90 min. The supernatant was aspirated, and RPMI-1640 medium was added again. After incubation at 37°C for 24 h, 500 μL of CFW staining solution was added, and the mixture was stained for 30 min. The supernatant was discarded, and biofilm formation was observed under a fluorescence microscope. The experiment was repeated three times.
[0043] Test results are as follows Figure 5 and Figure 6 As shown, Figure 5 The test results for strains SC5314 and 5172 are shown in the figure. Figure 6 The test results for strains C1, C2, and 12373 are shown in the figure.
[0044] Figure 5 The middle column shows the test results for FCCP (Fluidized Coptic Cytokinase) used in the left column, and the right column shows the test results for CFW (Fluidized Coptic Cytokinase) used in the right column. Figure 6 The middle column shows the test results for FCCP (Fluid Cholesterol) in the left column, and the right column shows the test results for CFW (Fluid Influenza) in the right column.
[0045] from Figure 5 and Figure 6 The results show that the CFW method was used to detect the effects of FCCP and Flu on the biofilm formation ability of Candida C1, C2, 12373, SC5314, and 5172. (The left side shows the quantitative fluorescence biofilm formation of Candida C1 treated with FCCP, and the right side shows the quantitative fluorescence biofilm formation of Candida C1 treated with Flu. From top to bottom, the biofilm formation of Candida C1, C2, 12372, SC5314, and 5172 is shown.) This indicates that, except for SC5314, FCCP has a stronger inhibitory effect on Candida C1 biofilm formation than Flu.
[0046] Example 4 The effect of FCCP on the mitochondrial morphology of Candida auris was detected using mitochondrial dyes. Test method: Adjust the concentration of each bacterial culture to 2×10⁻⁶ with PBS. 6 cells / mL. The bacterial concentration was adjusted to 2 × 10⁻⁶ cells / mL. 6 The FCCP concentrations in PBS were 0 μM and 40 μM per cell / mL. 2 mL of bacterial culture with different FCCP concentrations was placed in a confocal dish and incubated at 37°C for 90 min. The supernatant was aspirated, and PBS and 500 μL of mitochondrial staining solution were added again. After staining for 30 min, the supernatant was discarded, and the mitochondrial morphology was observed under a fluorescence microscope. The experiment was repeated three times.
[0047] The test results obtained are as follows Figure 7 and Figure 8 As shown, Figure 7 The results of mitochondrial dye testing for bacterial strains SC5314 and 5172 are shown in the figure. Figure 8 The results of mitochondrial dye testing for strains C1, C2, and 12373 are shown in the figure.
[0048] Figure 7 The middle column shows the test results with a drug dosage of 0 μM (left column), and the right column shows the test results with a drug dosage of 40 μM. Figure 8 The middle column shows the test results when the drug dosage was 0 μM, and the right column shows the test results when the drug dosage was 40 μM.
[0049] from Figure 7 and Figure 8 In the study, it was observed that, compared with the control group, the Candida albicans treated with 40 μM FCCP had a higher degree of mitochondrial fragmentation in most of the Candida albicans, forming fragmented punctate fluorescent structures or disconnected tubular structures that could not be connected, and were unable to form obvious and numerous tubular structures throughout the cells.
[0050] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.
Claims
1. Use of 4-trifluoromethoxyphenylhydrazone or its pharmaceutically acceptable salt as the sole active ingredient in any of the following: (i) A medicine for the preparation of a preventive or therapeutic drug for diseases caused by Candida infection, wherein the Candida is Candida albicans and / or Candida auris; (ii) Used to prepare antibacterial and / or bactericidal products for Candida, wherein the Candida is Candida albicans and / or Candida auris.
2. The use according to claim 1, characterized in that, The Candida species in question is wild-type Candida albicans SC5314.
3. The use according to claim 1, characterized in that, The minimum inhibitory concentration of the 4-trifluoromethoxyphenylhydrazone against wild-type Candida albicans SC5314 is 32 μg / mL.
4. The use according to claim 1, characterized in that, The Candida species in question is a drug-resistant strain of antifungal drugs.
5. The use according to claim 1, characterized in that, The Candida species mentioned is azole-resistant Candida albicans.
6. The use according to claim 1, characterized in that, The minimum inhibitory concentration of the 4-trifluoromethoxyphenylhydrazone-4-carbazide-resistant Candida albicans is 32 μg / mL.
7. The use according to claim 1, characterized in that, The Candida species is azole-resistant Candida auris, and the minimum inhibitory concentration of the azole-resistant Candida auris resistant to 4-trifluoromethoxyphenylhydrazone is 16 μg / mL.
8. The use according to claim 1, characterized in that, The drug is an inhibitor of Candida growth.
9. The use according to claim 1, characterized in that, The drug is a drug that inhibits the growth of Candida biofilm.
10. The use according to claim 1, characterized in that, The drug is a drug that destroys the mitochondria of Candida.