Application of combination of Cd1 and Mdr1 double-target inhibitor and azole drugs in preparation of antifungal drugs

Abstract

The invention relates to the technical field of biological medicine, in particular to application of a combination of a Cd1 and Mdr1 double-target inhibitor and an azole drug in preparation of an antifungal drug. Based on the fact that the compound as shown in the formula (I) can be used as a CCd1 and Mdr1 double-target inhibitor, the inhibition effect of the compound on candida albicans, candida auricula, candida glabrata, candida tropicalis, trichophyton rubrum and trichophyton indicus when the compound is combined with azole drugs is further evaluated, and the result shows that the compound as shown in the formula (I) has no obvious antibacterial activity on various fungi, and can be used for preparing a medicine for treating various fungi. The drug resistance of CCd1 and Mdr1 overexpression drug-resistant fungi can be reversed, the activity of Candida albicans, Candida auricula, Candida glabrata, Candida tropicalis, Trichophyton rubrum and Trichophyton indicus can be synergistically inhibited when the drug is combined with azole drugs, the treatment dosage of the azole drugs is effectively reduced, and the drug toxicity is small. Therefore, the compound shown in the formula (I) can improve the antifungal effect of azole drugs and reduce the drug resistance of the azole drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly to the use of a Cdr1 and Mdr1 dual-target inhibitor combined with an azole drug in the preparation of an antifungal drug. Background Art

[0002] In recent years, with the extensive use of broad-spectrum antibiotics, anti-tumor drugs and immunosuppressants, the widespread development of radiotherapy and organ transplantation, the widespread use of catheters and intubation, and the rapid increase in immunodeficiency patients, especially AIDS patients, the number of fungal infections, especially deep fungal infections, has increased significantly. Deep fungal infections have now become the main cause of death in patients with major diseases such as AIDS and tumors.

[0003] Azoles have become the first choice for the prevention and treatment of fungi due to their advantages such as few adverse effects and high bioavailability. However, due to the overuse of azoles, drug resistance is now very common, seriously affecting the therapeutic effect of azoles.

[0004] The ABC transporter Cdr1 and the MFS transporter Mdr1 are two efflux pumps that mediate fungal resistance to azoles. They belong to the ABC and MFS families, respectively. Mdr1 is proton-gradient driven and primarily effluxes azoles such as fluconazole. Cdr1, on the other hand, utilizes ATP hydrolysis to derive energy and can also efflux a wide range of azoles, contributing to multidrug resistance. Therefore, overexpression of Cdr1 and Mdr1 is a major mechanism of fungal resistance to azoles.

[0005] Therefore, the development of dual-target inhibitors of Cdr1 and Mdr1 can effectively enhance the sensitivity of fungi to azole drugs. Summary of the Invention

[0006] In order to overcome the above problems, the present invention provides the use of a Cdr1 and Mdr1 dual-target inhibitor combined with an azole drug in the preparation of an antifungal drug.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: The first aspect of the present invention provides the use of a compound represented by formula (I) in the preparation of a drug for enhancing the antifungal effect of azole drugs;

[0008] Formula (I).

[0009] In one or more embodiments, the azole drugs, including imidazole derivatives and triazole derivatives, are a class of drugs that exert antifungal effects by inhibiting the cytochrome P450-dependent enzyme-14α-lanolin alcohol demethylase (CYP51) to prevent the synthesis of ergosterol in fungal cell membranes.

[0010] Preferably, the azole drugs include one or more of clotrimazole, miconazole, econazole, fluconazole, ketoconazole, voriconazole, posaconazole and itraconazole.

[0011] In one or more embodiments, the mass ratio of the compound represented by formula (I) to the azole drug is (1-200):1, preferably (2-30):1, and more preferably (4-12):1.

[0012] In one or more embodiments, the fungus comprises one or more of Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida auris, Trichophyton rubrum and Trichophyton indica.

[0013] The second aspect of the present invention provides the use of a compound represented by formula (I) in the preparation of a drug for reducing resistance to azole drugs;

[0014] Formula (I).

[0015] In one or more embodiments, the azole drugs, including imidazole derivatives and triazole derivatives, are a class of drugs that exert antifungal effects by inhibiting the cytochrome P450-dependent enzyme-14α-lanolin alcohol demethylase (CYP51) to prevent the synthesis of ergosterol in fungal cell membranes.

[0016] Preferably, the azole drugs include one or more of clotrimazole, miconazole, econazole, fluconazole, ketoconazole, voriconazole, posaconazole and itraconazole.

[0017] In one or more embodiments, the mass ratio of the compound represented by formula (I) to the azole drug is (1-200):1, preferably (2-30):1, and more preferably (4-12):1.

[0018] The compound represented by formula (I) has an inhibitory effect on both Cdr1 and Mdr1 proteins, thus proving that the compound represented by formula (I) can serve as a dual-target inhibitor of Cdr1 and Mdr1; thereby, it can further reduce azole drug resistance and enhance the antifungal effect of azole drugs.

[0019] The third aspect of the present invention provides the use of a compound represented by formula (I) in combination with an azole drug in the preparation of an antifungal drug;

[0020] Formula (I).

[0021] In one or more embodiments, the azole drugs, including imidazole derivatives and triazole derivatives, are a class of drugs that exert antifungal effects by inhibiting the cytochrome P450-dependent enzyme-14α-lanolin alcohol demethylase (CYP51) to prevent the synthesis of ergosterol in fungal cell membranes.

[0022] Preferably, the azole drugs include one or more of clotrimazole, miconazole, econazole, fluconazole, ketoconazole, voriconazole, posaconazole and itraconazole.

[0023] In one or more embodiments, the mass ratio of the compound represented by formula (I) to the azole drug is (1-200):1, preferably (2-30):1, and more preferably (4-12):1.

[0024] In one or more embodiments, the fungus comprises one or more of Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida auris, Trichophyton rubrum and Trichophyton indica.

[0025] The fourth aspect of the present invention provides an antifungal drug comprising a compound represented by formula (I) and an azole drug;

[0026] Formula (I).

[0027] In one or more embodiments, the azole drugs, including imidazole derivatives and triazole derivatives, are a class of drugs that exert antifungal effects by inhibiting the cytochrome P450-dependent enzyme-14α-lanolin alcohol demethylase (CYP51) to prevent the synthesis of ergosterol in fungal cell membranes.

[0028] Preferably, the azole drugs include one or more of clotrimazole, miconazole, econazole, fluconazole, ketoconazole, voriconazole, posaconazole and itraconazole.

[0029] In one or more embodiments, the mass ratio of the compound represented by formula (I) to the azole drug is (1-200):1, preferably (2-30):1, and more preferably (4-12):1.

[0030] In one or more embodiments, the fungus comprises one or more of Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida auris, Trichophyton rubrum and Trichophyton indica.

[0031] In one or more embodiments, the medicine also includes pharmaceutically acceptable carriers, excipients and diluents etc. The non-pharmaceutical active ingredients such as the carriers, excipients and diluents that can be included are well known in the field, and those of ordinary skill in the art can determine that they meet clinical standards. Preferably, the carriers, excipients and diluents include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil etc.

[0032] Preferably, the dosage form of the drug is a suspension, emulsion, granule, spray, injection, transdermal absorbent, dosage form suitable for transfection, tablet, powder, granule or capsule.

[0033] The medicine of the present invention can be administered to the body in a known manner. For example, it can be delivered to the tissue of interest by systemic intravenous delivery or local injection. Alternatively, it can be administered intravenously, percutaneously, intranasally, through a mucosal membrane, or by other delivery methods. Such administration can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be administered in the present invention can vary depending on various factors to a great extent, such as the target cell, biological type or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0034] Preferably, the subjects of drug administration can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, orangutans, etc.

[0035] The fifth aspect of the present invention provides a pharmaceutical composition comprising the antifungal drug described in the fourth aspect.

[0036] The beneficial effects of the present invention are: (1) In the present invention, Mdr1-overexpressing drug-resistant Candida albicans G5 and Cdr1-overexpressing drug-resistant Candida albicans GU5 were selected as research objects, and the efflux pump substrate Rhodamine 6G (Rhodamine 6G) was used as an indicator to determine the effect of the compound represented by formula (I) on the efflux pump. The results showed that the addition of the compound represented by formula (I) had a significant efflux inhibitory effect in the experiment and was dose-dependent. At the same time, the flow cytometry experiment further confirmed that the compound represented by formula (I) had an inhibitory effect on the efflux pumps Cdr1 and Mdr1, thus proving that the compound represented by formula (I) can serve as a dual-target inhibitor of Cdr1 and Mdr1.

[0037] (2) Based on the fact that the compound represented by formula (I) can act as a dual-target inhibitor of Cdr1 and Mdr1, the inhibitory effect of the dual-target inhibitor of Cdr1 and Mdr1 in combination with azole drugs on Candida albicans, Candida auris, Candida glabrata, Candida tropicalis, Trichophyton rubrum, and Trichophyton indica was further evaluated. The results showed that the compound represented by formula (I) itself had no significant antifungal activity against various fungi, but it could reverse the drug resistance of Cdr1 and Mdr1-overexpressing drug-resistant fungi. In combination with azole drugs, it could synergistically inhibit the activity of Candida albicans, Candida auris, Candida glabrata, Candida tropicalis, Trichophyton rubrum, and Trichophyton indica, effectively reducing the therapeutic dose of azole drugs and having low drug toxicity. Therefore, the compound represented by formula (I) can enhance the antifungal effect of azole drugs and reduce azole drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0039] Figure 1 Figure 1 is a geometric mean (G-Mean value) graph of the flow cytometry experiment of CM-1 as a dual-target inhibitor, wherein A is the geometric mean graph of the uptake experiment of CM-1 using the efflux pump substrate Nile red dye on the GU5 strain, B is the geometric mean graph of the efflux experiment of CM-1 using the efflux pump substrate Nile red dye on the G5 strain, C is the geometric mean graph of the uptake experiment of CM-1 using the efflux pump substrate Rhodamine 6G dye on the GU5 strain, and D is the geometric mean graph of the efflux experiment of CM-1 using the efflux pump substrate Rhodamine 6G dye on the G5 strain; Figure 2 Figure 1 shows the validation results of CM-1 as a dual-target inhibitor, where A is the molecular docking diagram of CM-1 and Cdr1 protein; B is the result of the efflux experiment using rhodamine 6G dye on the GU5 strain, and C is the result of the absorption experiment using Nile red dye on the GU5 strain; D is the molecular docking diagram of CM-1 and Mdr1 protein; E is the result of the efflux experiment using rhodamine 6G dye on the G5 strain, and F is the result of the absorption experiment using Nile red dye on the G5 strain; Figure 3Figure 3 is the in vivo therapeutic effect of CM-1 combined with fluconazole on Candida albicans G5 and Candida albicans GU5 infection mouse models, wherein A is the survival curve of mice infected with Candida albicans G5 treated with CM-1 combined with fluconazole, B is the survival curve of mice infected with Candida albicans GU5 treated with CM-1 combined with fluconazole, C is the kidney bacterial load of mice infected with Candida albicans G5 treated with CM-1 combined with fluconazole, and D is the kidney bacterial load of mice infected with Candida albicans GU5 treated with CM-1 combined with fluconazole; in A to D, ns indicates no significant difference, * indicates p less than 0.05, ** indicates p less than 0.01, and *** indicates p less than 0.001; Figure 4 The results of kidney staining show the in vivo therapeutic effect of CM-1 combined with fluconazole on the mouse models infected with Candida albicans G5 and Candida albicans GU5. DETAILED DESCRIPTION

[0040] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0041] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0042] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0043] In the following examples, the compound represented by formula (I) is referred to as CM-1.

[0044] The drug-resistant Candida albicans G5 overexpressed Mdr1; the drug-resistant Candida albicans GU5 and drug-resistant Candida albicans DSY296 overexpressed Cdr1.

[0045] Candida albicans SC5314 was kindly donated by Northeastern University; Candida albicans G5, Candida albicans GU5, and Candida albicans DSY296 were kindly donated by the University of Würzburg, Germany; Candida tropicalis CT2 was obtained from Jinan Central Hospital; Candida auris CBS12774, Trichophyton rubrum, and Trichophyton indica were kindly donated by the Second Military Medical University of the Chinese People's Liberation Army.

[0046] Example 1 Rhodamine 6G efflux assay and Nile red uptake assay were used to evaluate the inhibitory effect on fungal efflux pump activity: (1) Rhodamine 6G efflux experiment: Candida albicans G5 or Candida albicans GU5 activated overnight were centrifuged and collected, washed once with PBS, and then placed in PBS and diluted to 2×10 6 CFU / mL; divided into experimental group, control group, untreated group and non-efflux group, the experimental group, control group, untreated group and non-efflux group were kept in a starvation state for 4 h, and the untreated group was not subjected to any subsequent operation; the experimental group, control group and non-efflux group were all added with rhodamine 6G (5 μM) and incubated at room temperature in the dark for 30 min, and washed three times with PBS to remove unbound pigment; CM-1 (4 or 8 μg / mL) was added to the suspension of the experimental group and incubated for 30 min; CM-1 was not added to the control group; 2% glucose was added to the experimental group and control group for incubation for 1 h to induce cell excretion; glucose was not added to the non-efflux group to induce cell excretion; then all samples from the experimental group, control group, untreated group and non-efflux group were collected, washed, and the intracellular fluorescence intensity of rhodamine 6G was measured by flow cytometry.

[0047] (2) Nile red uptake experiment: After overnight culture of Candida albicans G5 or Candida albicans GU5, the cells were centrifuged, washed with PBS, and then diluted to 2×10 6 The cells were divided into experimental, control and untreated groups. No subsequent operation was performed on the untreated group. Both the experimental and control groups were added with 5 μM Nile red dye. The experimental group was added with CM-1 (4 or 8 μg / mL) and incubated for 60 min. No CM-1 was added to the control group. The experimental, control and untreated groups were washed three times with PBS, and the Nile red fluorescence intensity was monitored by flow cytometry.

[0048] The results are as follows Figure 1 and Figure 2 As shown, in Figure 1 In Figures A and B, Nile red dye uptake experiments were performed using the Mdr1-overexpressing resistant strain G5 and the Cdr1-overexpressing resistant Candida albicans GU5, respectively. The untreated group showed only a very low background fluorescence signal; the control group showed a lower fluorescence intensity, demonstrating that when no inhibitor was added, the efflux channel was open and the amount of Nile red dye absorbed was small. In the 4 μg / mL CM-1 experimental group and the 8 μg / mL CM-1 experimental group, the fluorescence signal increased with increasing drug concentration, indicating that CM-1 promoted the intracellular accumulation of Nile red, and this effect was concentration-dependent. Figure 2Figures C and F also show that the fluorescence peak of the CM-1 experimental group shifted to the right, and the intracellular fluorescence intensity increased significantly, indicating that CM-1 promoted the accumulation of Nile red in cells, once again verifying the inhibitory effect of CM-1 on the efflux process.

[0049] Rhodamine 6G is a substrate of efflux pumps and can be used to evaluate the effects of compounds on strain efflux pumps. The Mdr1-overexpressing resistant strain G5 and the Cdr1-overexpressing resistant Candida albicans GU5 were selected as research objects, and the efflux pump substrate Rhodamine 6G was used as an indicator to determine the effect of CM-1 on the efflux pumps. Figure 1 Panels C and D in the middle show that the non-efflux group (serving as a positive control) had the highest fluorescence intensity, demonstrating intracellular accumulation of rhodamine 6G. The untreated group displayed only a very low background fluorescence signal. The control group exhibited a low fluorescence intensity, demonstrating that rhodamine 6G was completely effluxed in the absence of an inhibitor. However, treatment with 4 μg / mL CM-1 significantly enhanced intracellular fluorescence intensity. When the CM-1 concentration was increased to 8 μg / mL, the fluorescence intensity further increased, indicating that CM-1 inhibited the fungal efflux process in a dose-dependent manner. Figure 2 In Figures B and E, the untreated group also showed only basal fluorescence levels, and the control group showed a lower fluorescence intensity. However, after the addition of CM-1, the fluorescence peak shifted significantly to the right, indicating an increase in the accumulation of intracellular fluorescent substrates, further confirming that CM-1 can effectively inhibit efflux.

[0050] Example 2 The experimental strains growing in the logarithmic phase were collected by centrifugation and the culture medium was washed with PBS buffer. The OD value was measured by spectrophotometer to determine the cell density, and the cells were diluted to 1×10 3 Cells / mL, the diluted bacterial solution was used to prepare working solutions of azole drugs and a dual-target inhibitor of Cdr1 and Mdr1 (CM-1) with varying drug concentrations. In a 96-well plate, the horizontal rows of wells were filled with different concentrations of azole drug working solutions, ranging from 0.01 μg / mL to 256 μg / mL, and the vertical rows of wells were filled with different concentrations of the dual-target inhibitor of Cdr1 and Mdr1 (CM-1) working solutions, ranging from 0.25 μg / mL to 32 μg / mL. The plate was incubated in a 35°C incubator for 24 hours. The OD value of each well was measured at a wavelength of 600 nm. The control well was a blank well containing no drug. The growth rate of each well was calculated as the drug-treated well value / control well value × 100%. The growth rate of the control well was 100%. The drug concentration in the well where the absorbance decreased by 80% compared to the control well was determined as the minimum inhibitory concentration (MIC). 80 ).

[0051] Synergistic effect evaluation: Antimicrobial combinations can exhibit four types of effects in vitro or in vivo: "independent," "additive," "synergistic," and "antagonistic." The results of combined susceptibility testing are used to determine the interaction effect of two drugs when used in combination using the fractional inhibitory concentration index.

[0052] The fractional inhibitory concentration index (FICI) is calculated as follows: ΣFICI=FIC A +FIC B =C A / MIC A +C B / MIC B ; Among them, FICI is the fractional inhibitory concentration index, FIC A and FIC B The MIC values ​​of drugs A and B when used in combination are divided by the MIC values ​​of drugs A and B when used alone. A and MIC B are the minimum inhibitory concentrations of drugs A and B when used alone, C A with C B The concentration of each drug that achieves the same pharmacodynamic effect when used in combination. FICI > 4 indicates antagonism, FICI between 0.5 and 4 indicates additive or no effect, and FICI ≤ 0.5 is defined as synergistic.

[0053] The antifungal effects of CM-1 combined with azole drugs are shown in Tables 1 to 8.

[0054] Table 1 Antifungal effect of CM-1 combined with fluconazole

[0055] In Table 1, MIC: minimum inhibitory concentration; A : Minimum inhibitory concentration of CM-1 when the drug is used alone; C A : Minimum inhibitory concentration of CM-1 when drugs are used in combination; MIC B : Minimum inhibitory concentration of fluconazole when used alone; C B : minimum inhibitory concentration of fluconazole when the drugs are used in combination; FICI: fractional inhibitory concentration index; IN: combination index; SYN: synergistic effect.

[0056] Table 2 Antifungal effect of CM-1 combined with itraconazole

[0057] In Table 2, MIC: minimum inhibitory concentration; A : Minimum inhibitory concentration of CM-1 when the drug is used alone; C A: Minimum inhibitory concentration of CM-1 when drugs are used in combination; MIC B : Minimum inhibitory concentration of itraconazole when used alone; C B : minimum inhibitory concentration of itraconazole when the drugs are used in combination; FICI: fractional inhibitory concentration index; IN: combination index; SYN: synergistic effect.

[0058] Table 3 Antifungal effect of CM-1 combined with voriconazole

[0059] In Table 3, MIC: minimum inhibitory concentration; A : Minimum inhibitory concentration of CM-1 when the drug is used alone; C A : Minimum inhibitory concentration of CM-1 when drugs are used in combination; MIC B : Minimum inhibitory concentration of voriconazole when used alone; C B : minimum inhibitory concentration of voriconazole when the drugs are used in combination; FICI: fractional inhibitory concentration index; IN: combination index; SYN: synergistic effect.

[0060] Table 4 Antifungal effect of CM-1 combined with posaconazole

[0061] In Table 4, MIC: minimum inhibitory concentration; A : Minimum inhibitory concentration of CM-1 when the drug is used alone; C A : Minimum inhibitory concentration of CM-1 when drugs are used in combination; MIC B : Minimum inhibitory concentration of posaconazole when used alone; C B : minimum inhibitory concentration of posaconazole when the drugs are used in combination; FICI: fractional inhibitory concentration index; IN: combination index; SYN: synergistic effect.

[0062] Table 5 Antifungal effect of CM-1 combined with clotrimazole

[0063] In Table 5, MIC: minimum inhibitory concentration; A : Minimum inhibitory concentration of CM-1 when the drug is used alone; C A : Minimum inhibitory concentration of CM-1 when drugs are used in combination; MIC B : Minimum inhibitory concentration of clotrimazole when the drug is used alone; C B : minimum inhibitory concentration of clotrimazole when the drugs are used in combination; FICI: fractional inhibitory concentration index; IN: combination index; SYN: synergistic effect.

[0064] Table 6 Antifungal effect of CM-1 combined with miconazole

[0065] In Table 6, MIC: minimum inhibitory concentration; A : Minimum inhibitory concentration of CM-1 when the drug is used alone; C A : Minimum inhibitory concentration of CM-1 when drugs are used in combination; MIC B : Minimum inhibitory concentration of miconazole when used alone; C B : minimum inhibitory concentration of miconazole when the drugs are used in combination; FICI: fractional inhibitory concentration index; IN: combination index; SYN: synergistic effect.

[0066] Table 7 Antifungal effect of CM-1 combined with ketoconazole

[0067] In Table 7, MIC: minimum inhibitory concentration; A : Minimum inhibitory concentration of CM-1 when the drug is used alone; C A : Minimum inhibitory concentration of CM-1 when drugs are used in combination; MIC B : Minimum inhibitory concentration of ketoconazole when used alone; C B : minimum inhibitory concentration of ketoconazole when the drugs are used in combination; FICI: fractional inhibitory concentration index; IN: combination index; SYN: synergistic effect.

[0068] Table 8 Antifungal effect of CM-1 combined with econazole

[0069] In Table 8, MIC: minimum inhibitory concentration; A : Minimum inhibitory concentration of CM-1 when the drug is used alone; C A : Minimum inhibitory concentration of CM-1 when drugs are used in combination; MIC B : Minimum inhibitory concentration of econazole when used alone; C B : minimum inhibitory concentration of econazole when the drugs are used in combination; FICI: fractional inhibitory concentration index; IN: combination index; SYN: synergistic effect.

[0070] From the results in Tables 1 to 8, it can be seen that CM-1 itself has no antibacterial activity against Candida albicans, Candida auris, Candida glabrata, Candida tropicalis, Trichophyton rubrum, and Trichophyton indica, but can reverse the drug resistance of Cdr1- and Mdr1-overexpressing drug-resistant fungi. Combination with azole drugs can synergistically inhibit the activity of Candida albicans, Candida auris, Candida glabrata, Candida tropicalis, Trichophyton rubrum, and Trichophyton indica, effectively reducing the therapeutic dose of azole drugs and having low drug toxicity.

[0071] Example 3 Cytotoxicity test of CM-1: Cell lines: HEK293 human embryonic kidney cells, BEAS-2B human bronchial epithelial cells, and HUVEC human umbilical artery epithelial cells.

[0072] CM-1 was dissolved in dimethyl sulfoxide (DMSO) to a 10 mM stock solution.

[0073] The cells in the logarithmic growth phase were digested with trypsin and the cell suspension was collected in a sterile centrifuge tube. The cell density was adjusted to 5×10 4 Cells were seeded at 100 μg / mL in 96-well plates and incubated overnight to allow attachment. The supernatant was replaced with fresh RPMI-1640 medium containing varying concentrations of CM-1. No CM-1 was added as a negative control, and the 96-well plates were incubated in a 37°C, 5% CO2 incubator for 24 hours. After incubation, cell morphology was observed, and 10 μL of 5 mg / mL thiazolyl blue (MTT) solution was added to each well. After 4 hours, the supernatant was discarded, and 100 μL of DMSO was added to each well, mixed thoroughly, and absorbance at 490 nm was measured using a microplate reader.

[0074] The results are shown in Table 9, which showed that CM-1 exhibited good low cytotoxicity to HEK293, RAW264.7, and HUVEC.

[0075] Table 9 Cytotoxicity test results

[0076] Example 4 In vivo antibacterial validation: In the monotherapy and combination therapy experiments, 6- to 8-week-old male BALB / c mice were randomly divided into four groups (n=10 per group): CM-1 monotherapy group, fluconazole monotherapy group, CM-1 combined with fluconazole therapy group, and saline control group. Overnight activated Candida albicans GU5 or Candida albicans G5 yeast phase cells were adjusted to a concentration of 1×10 7Each mouse was injected with 100 μL of bacterial suspension via the tail vein. Twenty-four hours after infection, dosing was initiated: the CM-1 monotherapy group received an intraperitoneal injection of 20 mg / kg CM-1, the fluconazole monotherapy group received 5 mg / kg fluconazole, and the CM-1 combined with fluconazole group received simultaneous injections of 20 mg / kg CM-1 and 5 mg / kg fluconazole. The control group received an equal volume of sterile saline. Dosing was performed once daily for three consecutive days. Survival rates were recorded daily during treatment. On the fourth day, mice were humanely sacrificed and bilateral kidneys were harvested. The left kidney was fixed with 4% paraformaldehyde for histopathological analysis. The fixed tissue was embedded in paraffin and sectioned for histomorphological observation and assessment of fungal infiltration using PAS and hematoxylin and eosin (H&E) staining. The right kidney was used for real-time measurement of renal fungal burden.

[0077] The results are as follows Figure 3 and Figure 4 As shown, the survival curves of the groups treated with either CM-1 or fluconazole alone did not differ significantly from those in the PBS group. However, the survival rate in the group treated with CM-1 combined with fluconazole was significantly improved compared with the monotherapy group. Analysis of renal fungal burden showed a significant reduction in fungal pathogenicity in the group treated with CM-1 combined with fluconazole. Histopathological examination revealed that Candida albicans was rarely found in the kidneys of the group treated with CM-1 combined with fluconazole, and renal inflammatory cell infiltration was significantly reduced. These findings suggest that the combination of CM-1 and fluconazole has significant clinical potential as a novel strategy for combating drug-resistant fungal infections.

[0078] Among them, in the Candida albicans G5 group, the bacterial load of the control group was 2.84×10 8 CFU / g, and the bacterial load in the CM-1 monotherapy group was 4.66×10 8 CFU / g, and the load in the fluconazole monotherapy group was 1.27×10 8 CFU / g, and the bacterial load in the CM-1 combined with fluconazole treatment group was 4.75×10 7 CFU / g; In the Candida albicans GU5 group, the bacterial load of the control group was 3.40×10 8 CFU / g, and the bacterial load in the CM-1 monotherapy group was 3.83×10 8 CFU / g, and the load in the fluconazole monotherapy group was 4.09×10 8 CFU / g, and the bacterial load in the CM-1 combined with fluconazole treatment group was 1.60×10 7 CFU / g.

[0079] The bacterial load of each group was the average value.

[0080] Example 5 Compared with Example 4, the drug ratio of CM-1 to fluconazole was adjusted, and the other experimental procedures were the same as in Example 4. Specifically, the CM-1 monotherapy group was intraperitoneally injected with 20 mg / kg CM-1, the fluconazole monotherapy group was injected with 3 mg / kg fluconazole, the CM-1 combined with fluconazole treatment group was simultaneously injected with 20 mg / kg CM-1 and 3 mg / kg fluconazole, and the control group was injected with an equal volume of sterile saline.

[0081] The results are shown in Table 10. As can be seen from the table, the fungal burden analysis of the kidneys showed that the fungal pathogenicity of the CM-1 combined with fluconazole treatment group was significantly reduced. However, compared with the simultaneous injection of 20 mg / kg CM-1 and 5 mg / kg fluconazole in Example 4, the fungal burden of the kidneys infected with Candida albicans GU5 increased. These findings indicate that the CM-1 and fluconazole combination treatment regimen is related to the dosage of the drugs used.

[0082] Table 10 Verification of the combined effect of CM-1 and fluconazole in the mouse systemic infection model

[0083] Example 6 Compared with Example 4, the drug ratio of CM-1 to fluconazole was adjusted, and the other experimental procedures were the same as in Example 4. Specifically, the CM-1 monotherapy group was intraperitoneally injected with 10 mg / kg CM-1, the fluconazole monotherapy group was injected with 7 mg / kg fluconazole, the CM-1 combined with fluconazole treatment group was simultaneously injected with 10 mg / kg CM-1 and 7 mg / kg fluconazole, and the control group was injected with an equal volume of sterile saline.

[0084] The results are shown in Table 11. As can be seen from the table, the fungal burden analysis of the kidneys showed that the fungal pathogenicity of the CM-1 combined with fluconazole treatment group was significantly reduced. However, compared with the simultaneous injection of 20 mg / kg CM-1 and 5 mg / kg fluconazole in Example 4, the fungal burden of the kidneys increased. These findings indicate that the CM-1 and fluconazole combination treatment regimen is related to the dosage of the drugs used.

[0085] Table 11 Verification of the combined effect of CM-1 and fluconazole in mouse systemic infection model

[0086] Example 7 Compared with Example 4, the drug ratio of CM-1 to fluconazole was adjusted, and the other experimental procedures were the same as in Example 4. Specifically, the CM-1 monotherapy group was intraperitoneally injected with 6 mg / kg CM-1, the fluconazole monotherapy group was injected with 6 mg / kg fluconazole, the CM-1 combined with fluconazole treatment group was simultaneously injected with 6 mg / kg CM-1 and 6 mg / kg fluconazole, and the control group was injected with an equal volume of sterile saline.

[0087] The results are shown in Table 12. As can be seen from the table, the fungal burden analysis of the kidneys showed that the fungal pathogenicity of the CM-1 combined with fluconazole treatment group was significantly reduced. However, compared with the simultaneous injection of 20 mg / kg CM-1 and 5 mg / kg fluconazole in Example 4, the fungal burden of the kidneys increased. These findings indicate that the CM-1 and fluconazole combination treatment regimen is related to the dosage of the drugs used.

[0088] Table 12 Validation of the combined effect of CM-1 and fluconazole in a mouse systemic infection model

[0089] Example 8 Compared with Example 4, the drug ratio of CM-1 to fluconazole was adjusted, and the other experimental procedures were the same as in Example 4. Specifically, the CM-1 monotherapy group was intraperitoneally injected with 60 mg / kg CM-1, the fluconazole monotherapy group was injected with 3 mg / kg fluconazole, the CM-1 combined with fluconazole treatment group was simultaneously injected with 60 mg / kg CM-1 and 3 mg / kg fluconazole, and the control group was injected with an equal volume of sterile saline.

[0090] The results are shown in Table 13. As can be seen from the table, the fungal burden analysis of the kidneys showed that the fungal pathogenicity of the CM-1 combined with fluconazole treatment group was significantly reduced. However, compared with the simultaneous injection of 20 mg / kg CM-1 and 5 mg / kg fluconazole in Example 4, the fungal burden of the kidneys increased. These findings indicate that the CM-1 and fluconazole combination treatment regimen is related to the dosage of the drugs used.

[0091] Table 13 Validation of the combined effect of CM-1 and fluconazole in a mouse systemic infection model

[0092] Example 9 Compared with Example 4, the species of fungi used were adjusted to be Candida auris CBS12774, Candida glabrata CG2, and Candida tropicalis CT2, and the other experimental steps were the same as those in Example 4.

[0093] The results are shown in Table 14. In mouse models infected with Candida auris CBS12774, Candida glabrata CG2, and Candida tropicalis CT2, the bacterial load in the CM-1 alone group was not significantly different from that in the control group. However, analysis of renal fungal load in the combination treatment group revealed a significant reduction in fungal pathogenicity. These findings suggest that the CM-1 and fluconazole combination therapy has significant potential for the treatment of multidrug-resistant bacterial infections.

[0094] Table 14 Validation of the combined effect of CM-1 and fluconazole in a mouse systemic infection model

[0095] Example 10 Validation of the topical combination of CM-1 and fluconazole against Trichophyton rubrum and Trichophyton indica in a mouse skin infection model: Male BALB / c mice weighing 20-22 g were randomly housed in individual cages with four mice per cage and acclimatized for 3 days before the experiment. 50 mg kg -1 Cyclophosphamide was used to induce neutropenia; subsequently, 50 mg kg -1 Mice were anesthetized with sodium pentobarbital and full-thickness skin perforations were performed on the back skin using a 0.8 cm diameter biopsy punch. Suspensions of Trichophyton rubrum and Trichophyton indica (1 × 10 8 CFU / mL, 50 μL per mouse) was inoculated into the circular wound until the skin appeared moist but without excess fluid. 24 h after inoculation, approximately 30 mg of different ointments, including 2% (mass fraction) CM-1 ointment, 1% (mass fraction) fluconazole ointment, and 2% (mass fraction) CM-1 and 1% (mass fraction) fluconazole ointment, were applied to the infected skin area and covered with a circular paraffin film. At the same time, an ointment containing only matrix was used as a negative control. All ointments were applied at intervals of 12 h for a total of 3 times. Fungal counts in wound specimens were recorded on the 8th day after infection.

[0096] The results are shown in Table 15. In the mouse models of T. rubrum and T. indica infection, the bacterial load in the CM-1 alone group was not significantly different from that in the control group; however, the fungal pathogenicity was significantly reduced in the combination treatment group. These findings suggest that the CM-1 and fluconazole combination therapy has significant potential for the treatment of multidrug-resistant bacterial infections.

[0097] Table 15: The combined effect of CM-1 and fluconazole against Trichophyton rubrum and Trichophyton indica verified by mouse skin infection model

[0098] Example 11 Validation of the topical combination of CM-1 and clotrimazole in a mouse skin infection model: Compared with Example 10, the modified fungal species were Candida albicans GU5, Candida auris CBS12774, Candida glabrata CG2, Candida tropicalis CT2, Trichophyton rubrum, and Trichophyton indica; after successfully establishing a mouse skin infection model infected with different fungi, approximately 30 mg of different ointments were applied to the infected skin, including an ointment containing 2% (mass fraction) CM-1, an ointment containing 1% (mass fraction) clotrimazole, and an ointment containing both 2% (mass fraction) CM-1 and 1% (mass fraction) clotrimazole, and covered with a circular paraffin film; the other experimental procedures were the same as in Example 10.

[0099] The results are shown in Table 16.

[0100] Table 16: Mouse skin infection model to verify the effect of combined external use of CM-1 and clotrimazole

[0101] Example 12 Mouse skin infection model verifies the effect of topical combination of CM-1 and miconazole: Compared with Example 11, the azole drug in Example 11 was changed to miconazole, and other methods were the same as those in Example 11.

[0102] The results are shown in Table 17.

[0103] Table 17: Effect of combined topical application of CM-1 and miconazole verified in a mouse skin infection model

[0104] Example 13 Mouse skin infection model verifies the effect of combined topical application of CM-1 and ketoconazole: Compared with Example 11, the azole drug in Example 11 was changed to ketoconazole, and the other methods were the same as those in Example 11.

[0105] The results are shown in Table 18.

[0106] Table 18: Effect of combined topical application of CM-1 and ketoconazole verified in a mouse skin infection model

[0107] Example 14 Mouse skin infection model verifies the effect of topical combination of CM-1 and econazole: Compared with Example 11, the azole drug in Example 11 was changed to econazole, and other methods were the same as those in Example 11.

[0108] The results are shown in Table 19.

[0109] Table 19: Effect of combined topical application of CM-1 and econazole verified in a mouse skin infection model

[0110] The results in Tables 16-19 demonstrate that CM-1 exhibits significant synergistic effects when combined with clotrimazole, miconazole, ketoconazole, and econazole in the treatment of fungal skin infections in mice, rapidly clearing pathogens, alleviating tissue inflammation, and promoting skin healing. This study provides a novel approach to overcoming azole resistance, not only demonstrating the significant potential of CM-1 as an antifungal potentiator but also laying a solid theoretical and experimental foundation for the development of more effective and safer combination therapies for superficial fungal infections, demonstrating its significant clinical translational potential.

[0111] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of the compound represented by formula (I) in the preparation of a drug for enhancing the antifungal effect of azole drugs; Formula (I).

2. Use of the compound represented by formula (I) in the preparation of drugs for reducing resistance to azole drugs; Formula (I).

3. Use of the compound represented by formula (I) in combination with azole drugs in the preparation of antifungal drugs; Formula (I).

4. The use according to any one of claims 1 to 3, wherein The azole drugs, including imidazole derivatives and triazole derivatives, are a class of drugs that exert antifungal effects by inhibiting the cytochrome P450-dependent enzyme-14α-lanolin alcohol demethylase to prevent the synthesis of ergosterol in fungal cell membranes.

5. The use according to claim 4, characterized in that The azole drugs include one or more of clotrimazole, miconazole, econazole, fluconazole, ketoconazole, voriconazole, posaconazole and itraconazole; The mass ratio of the compound represented by formula (I) to the azole drug is (1~200):

1.

6. The use according to claim 4, characterized in that The fungi include one or more of Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida auris, Trichophyton rubrum and Trichophyton indica.

7. An antifungal drug, characterized in that: It includes a compound represented by formula (I) and an azole drug; Formula (I).

8. The antifungal drug according to claim 7, wherein The azole drugs, including imidazole derivatives and triazole derivatives, are a class of drugs that exert antifungal effects by inhibiting the cytochrome P450-dependent enzyme-14α-lanolin alcohol demethylase to prevent the synthesis of ergosterol in fungal cell membranes.

9. The antifungal drug according to claim 8, wherein The azole drugs include one or more of clotrimazole, miconazole, econazole, fluconazole, ketoconazole, voriconazole, posaconazole and itraconazole; The mass ratio of the compound represented by formula (I) to the azole drug is (1-200):1; The fungi include one or more of Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida auris, Trichophyton rubrum and Trichophyton indica.

10. A pharmaceutical composition, characterized in that The antifungal drug according to any one of claims 7 to 9.

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