Use of a dual-target inhibitor of cdr1 and mdr1 in combination with an azole in the preparation of an antifungal drug

Abstract

The present application relates to the technical field of biological medicine, in particular to the application of Cdr1 and Mdr1 double-target inhibitor combined with azole drugs in the preparation of antifungal drugs. Based on the compound shown in formula (I) can be used as Cdr1 and Mdr1 double-target inhibitor, the inhibitory effect of the compound on Candida albicans, Candida auris, Candida glabrata, Candida tropicalis, Trichophyton rubrum and Trichophyton indiana combined with azole drugs was further evaluated. The results show that the compound shown in formula (I) itself has no obvious antibacterial activity on various fungi, but can reverse the drug resistance of Cdr1 and Mdr1 overexpression drug-resistant fungi. The combination of the compound with azole drugs can synergistically inhibit the activity of Candida albicans, Candida auris, Candida glabrata, Candida tropicalis, Trichophyton rubrum and Trichophyton indiana, effectively reduce the therapeutic dose of azole drugs, and the drug toxicity is small. Therefore, the compound shown in formula (I) can improve the antifungal effect of azole drugs and reduce the drug resistance of azole drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the application of Cdr1 and Mdr1 double-target inhibitor combined with azole drugs in the preparation of antifungal drugs. BACKGROUND

[0002] In recent years, with the extensive use of broad-spectrum antibiotics, antitumor drugs and immunosuppressive agents, the widespread development of radiotherapy and organ transplantation, the universal development of catheters and cannulas, and the rapid increase of immunodeficient patients, especially AIDS patients, the number of fungal infections, especially deep fungal infections, has increased significantly. Deep fungal infection has become a major cause of death in patients with AIDS and cancer and other major diseases.

[0003] Azole drugs have become the first choice for the prevention and treatment of fungi due to their small adverse effects and high bioavailability. However, due to the overuse of azole drugs, drug resistance has become very common, which seriously affects the therapeutic effect of azole drugs.

[0004] ABC transporter Cdr1 and MFS transporter Mdr1 are two kinds of efflux pumps that mediate fungal resistance to azole drugs, belonging to ABC and MFS families, respectively. Mdr1 depends on proton gradient driving, mainly expelling azole drugs such as fluconazole; while Cdr1 is powered by ATP hydrolysis, which can also expel a variety of azole drugs, leading to multidrug resistance. Therefore, the overexpression of Cdr1 and Mdr1 is one of the main mechanisms of fungal resistance to azole drugs.

[0005] Therefore, the development of Cdr1 and Mdr1 double-target inhibitors can effectively improve the sensitivity of fungi to azole drugs. SUMMARY

[0006] In order to overcome the above problems, the present application provides the application of Cdr1 and Mdr1 double-target inhibitor combined with azole drugs in the preparation of antifungal drugs.

[0007] To achieve the above technical purposes, the technical scheme adopted by the present application is as follows:

[0008] In a first aspect of the present application, the application of the compound shown in formula (I) in the preparation of a drug for improving the antifungal effect of azole drugs is provided.

[0009]

[0010] Formula (I).

[0011] 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 cytochrome P450-dependent enzyme-14 alpha-lanosterol demethylase (CYP51) to prevent the synthesis of ergosterol in the fungal cell membrane.

[0012] Preferably, the azole drugs include one or more of clotrimazole, miconazole, econazole, fluconazole, ketoconazole, voriconazole, posaconazole and itraconazole.

[0013] In one or more embodiments, the mass ratio of the compound of formula (I) to the azole drug is (1-200):1, preferably (2-30):1, and further preferably (4-12):1.

[0014] In one or more embodiments, the fungi include one or more of Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida auris, Trichophyton rubrum and Trichophyton indiana.

[0015] In a second aspect of the present application, the use of the compound of formula (I) in the preparation of a drug for reducing azole drug resistance is provided.

[0016]

[0017] Formula (I).

[0018] 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 cytochrome P450-dependent enzyme-14 alpha-lanosterol demethylase (CYP51) to prevent the synthesis of ergosterol in the fungal cell membrane.

[0019] Preferably, the azole drugs include one or more of clotrimazole, miconazole, econazole, fluconazole, ketoconazole, voriconazole, posaconazole and itraconazole.

[0020] In one or more embodiments, the mass ratio of the compound of formula (I) to the azole drug is (1-200):1, preferably (2-30):1, and further preferably (4-12):1.

[0021] The compound of formula (I) has inhibitory effects on both Cdr1 and Mdr1 proteins, thus proving that the compound of formula (I) can act as a Cdr1 and Mdr1 dual-target inhibitor; and can further reduce azole drug resistance and improve the antifungal effects of azole drugs.

[0022] In a third aspect of the present application, the use of the compound of formula (I) in combination with azole drugs in the preparation of an antifungal drug is provided.

[0023]

[0024] Formula (I).

[0025] In one or more embodiments, the azole drug, including imidazole derivatives and triazole derivatives, is a class of drugs that exerts antifungal effect by inhibiting cytochrome P450-dependent enzyme-14α-lanosterol demethylase (CYP51) to prevent the synthesis of ergosterol in the fungal cell membrane.

[0026] Preferably, the azole drug includes one or more of clotrimazole, miconazole, econazole, fluconazole, ketoconazole, voriconazole, posaconazole and itraconazole.

[0027] In one or more embodiments, the mass ratio of the compound of Formula (I) to the azole drug is (1-200):1, preferably (2-30):1, and further preferably (4-12):1.

[0028] In one or more embodiments, the fungus includes one or more of Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida auris, Trichophyton rubrum and Trichophyton indiana.

[0029] In a fourth aspect of the present application, an antifungal drug is provided, including a compound of Formula (I) and an azole drug;

[0030]

[0031] Formula (I).

[0032] In one or more embodiments, the azole drug, including imidazole derivatives and triazole derivatives, is a class of drugs that exerts antifungal effect by inhibiting cytochrome P450-dependent enzyme-14α-lanosterol demethylase (CYP51) to prevent the synthesis of ergosterol in the fungal cell membrane.

[0033] Preferably, the azole drug includes one or more of clotrimazole, miconazole, econazole, fluconazole, ketoconazole, voriconazole, posaconazole and itraconazole.

[0034] In one or more embodiments, the mass ratio of the compound of Formula (I) to the azole drug is (1-200):1, preferably (2-30):1, and further preferably (4-12):1.

[0035] In one or more embodiments, the fungus includes one or more of Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida auris, Trichophyton rubrum and Trichophyton indiana.

[0036] In one or more embodiments, the medicament further comprises pharmaceutically acceptable carriers, excipients and diluents, etc. The non-drug active ingredients that can be contained in the carriers, excipients and diluents, etc. are well known in the art, and those skilled in the art can determine whether they meet the 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, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, etc.

[0037] Preferably, the dosage form of the medicament is a suspension, an emulsion, a granule, a spray, an injection, a transdermal absorption agent, a suitable transfection agent, a tablet, a powder, a granule or a capsule.

[0038] The medicament of the present application can be administered into the body by known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Alternatively, administration is via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration can be via single or multiple doses. The skilled person understands that the actual dose to be administered in the present application can vary greatly depending on a variety of factors, such as the target cell, the biological type or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

[0039] Preferably, the administration subject of the medicament can be human and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, orangutans, etc.

[0040] In a fifth aspect of the present application, a pharmaceutical composition is provided, comprising the antifungal medicament of the fourth aspect.

[0041] The present application has the following beneficial effects:

[0042] (1) In the present application, Mdr1 overexpression resistant Candida albicans G5 and Cdr1 overexpression resistant Candida albicans GU5 are selected as the research object, and the efflux pump substrate Rhodamine 6G is used as an indicator to judge the effect of the compound represented by formula (I) on the efflux pump. The results show that the addition of the compound represented by formula (I) in the experiment has obvious efflux inhibition and has a dose-dependent effect. At the same time, the flow experiment further confirms that the compound represented by formula (I) has an inhibitory effect on the efflux pumps Cdr1 and Mdr1, thus proving that the compound represented by formula (I) can be used as a Cdr1 and Mdr1 dual-target inhibitor.

[0043] (2) Based on the compound shown in formula (I) can be used as Cdr1 and Mdr1 dual-target inhibitor, further evaluated the Cdr1 and Mdr1 dual-target inhibitor and azole drugs on Candida albicans, Candida auris, Candida glabrata, Candida tropicalis, Trichophyton rubrum and Trichophyton indiana inhibition effect, the results show that the compound shown in formula (I) itself has no obvious antibacterial activity on various fungi, but can reverse the drug resistance of Cdr1 and Mdr1 overexpression resistant fungi, and the combination of azole drugs can synergistically inhibit the activity of Candida albicans, Candida auris, Candida glabrata, Candida tropicalis, Trichophyton rubrum and Trichophyton indiana, effectively reduce the therapeutic dose of azole drugs, and the drug toxicity is small. Therefore, the compound shown in formula (I) can improve the antifungal effect of azole drugs and reduce the drug resistance of azole drugs. BRIEF DESCRIPTION OF DRAWINGS

[0044] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The embodiments of the present application, and their

[0045] Figure 1 The figure is the geometric mean value (G-Mean value) graph of the flow experiment of CM-1 as a dual-target inhibitor, wherein A is the geometric mean value graph of the absorption experiment of CM-1 using the efflux pump substrate Nile red dye to test GU5 strain, B is the geometric mean value graph of the efflux experiment of CM-1 using the efflux pump substrate Nile red dye to test G5 strain, C is the geometric mean value graph of the absorption experiment of CM-1 using the efflux pump substrate rhodamine 6G dye to test GU5 strain, D is the geometric mean value graph of the efflux experiment of CM-1 using the efflux pump substrate rhodamine 6G dye to test G5 strain;

[0046] Figure 2 The figure is the verification result graph of CM-1 as a dual-target inhibitor, wherein A is the molecular docking graph of CM-1 and Cdr1 protein; B is the efflux experiment result graph of GU5 strain using rhodamine 6G dye, C is the absorption experiment result graph of GU5 strain using Nile red dye; D is the molecular docking graph of CM-1 and Mdr1 protein; E is the efflux experiment result graph of G5 strain using rhodamine 6G dye, F is the absorption experiment result graph of G5 strain using Nile red dye;

[0047] Figure 3Figures for the in vivo therapeutic effect of CM-1 combined with fluconazole on the mouse models infected with Candida albicans G5 and Candida albicans GU5, wherein A is the survival curve of the mouse infected with Candida albicans G5 treated by CM-1 combined with fluconazole, B is the survival curve of the mouse infected with Candida albicans GU5 treated by CM-1 combined with fluconazole, C is the kidney fungal load of the mouse infected with Candida albicans G5 treated by CM-1 combined with fluconazole, and D is the kidney fungal load of the mouse infected with Candida albicans GU5 treated by CM-1 combined with fluconazole; in A-D, ns represents no significant difference, * represents p less than 0.05, ** represents p less than 0.01, and *** represents p less than 0.001.

[0048] Figure 4 Figures for the kidney staining results of 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

[0049] It should be noted that the following detailed description is merely exemplary in nature and is intended to provide further description of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0050] It is also important to note that the terms "comprises", "comprising", "includes", "including", "contains", "containing" or variations thereof herein, do not specify an exhaustive or complete list of elements or steps as they can be subject to revision by the skilled person depending on the specific application.

[0051] In order to enable persons skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with specific examples.

[0052] In the following examples, the compound represented by formula (I) is referred to as CM-1.

[0053] Drug-resistant Candida albicans G5 overexpresses Mdr1; drug-resistant Candida albicans GU5 and drug-resistant Candida albicans DSY296 overexpress Cdr1.

[0054] Candida albicans SC5314 was donated by Northeastern University, USA; Candida albicans G5, Candida albicans GU5 and Candida albicans DSY296 were donated by University of Wurzburg, Germany; Candida tropicalis CT2 was derived from Jinan Central Hospital; Candida auris CBS12774, Trichophyton rubrum and Trichophyton indicum were donated by the Second Military Medical University of the People's Liberation Army.

[0055] Example 1

[0056] Rhodamine 6G efflux experiment and Nile red uptake experiment were used to evaluate the inhibitory effect of fungal efflux pump activity:

[0057] (1) Rhodamine 6G efflux experiment: centrifugal collection of overnight activated white Candida G5 or white Candida GU5, washed once with PBS and then placed in PBS, diluted to 2 x 10 6 CFU / mL; divide the experimental group, the control group, the untreated group and the non-efflux group, and maintain the experimental group, the control group, the untreated group and the non-efflux group in a starved state for 4 h, and the untreated group is not operated subsequently; the experimental group, the control group and the non-efflux group are added with Rhodamine 6G (5 μM) and placed at room temperature, avoiding light for 30 min, washed with PBS for 3 times to remove the unbound pigment; CM-1 (4 or 8 μg / mL) is added to the suspension of the experimental group for incubation for 30 min; the control group is not added with CM-1; the experimental group and the control group are added with 2% glucose for incubation for 1 h to make them produce cell discharge; the non-efflux group is not added with glucose to make it not produce cell discharge; then all samples of the experimental group, the control group, the untreated group and the non-efflux group are collected, washed and the fluorescence intensity of Rhodamine 6G in the cells is determined by flow cytometry.

[0058] (2) Nile red uptake experiment: centrifugal collection of overnight cultured white Candida G5 or white Candida GU5, washed with PBS, then diluted to 2 x 10 6 CFU / mL with 2% glucose PBS buffer, and divided into experimental group, control group and untreated group, wherein the untreated group is not operated subsequently; the experimental group and the control group are added with 5 μM Nile red dye, and the experimental group is added with CM-1 (4 or 8 μg / mL) for incubation for 60 min; the control group is not added with CM-1, and the experimental group, the control group and the untreated group are washed with PBS for 3 times, and the Nile red fluorescence intensity is monitored by flow cytometry.

[0059] The results are shown in Figure 1 and Figure 2 , in the A and B graphs of Figure 1 , Mdr1 overexpression drug-resistant strain G5 and Cdr1 overexpression drug-resistant white Candida GU5 were used for Nile red dye uptake experiment; the untreated group only showed very low background fluorescence signal; the control group showed low fluorescence intensity, proving that when no inhibitor was added, the efflux channel was open and the Nile red dye absorption was low; the fluorescence signal in the 4 μg / mL CM-1 experimental group and the 8 μg / mL CM-1 experimental group increased with the increase of drug concentration, indicating that CM-1 promoted the accumulation of Nile red in the cells, and the effect was concentration-dependent. Figure 2Fig. 2C and Fig. 2F also show that the fluorescence peak of the CM-1 experimental group is right-shifted, and the intracellular fluorescence intensity is significantly improved, indicating that CM-1 promotes the accumulation of Nile red in the cell, and again verifies the inhibitory effect of CM-1 on the efflux process.

[0060] Rhodamine 6G is a substrate of efflux pump, which can be used to evaluate the effect of compounds on the efflux pump of the strain. Mdr1 overexpressing drug-resistant strain G5 and Cdr1 overexpressing drug-resistant Candida albicans GU5 were selected as research objects, and efflux pump substrate rhodamine 6G was used as an indicator to determine the effect of CM-1 on the efflux pump. Figure 1 Fig. 2C and Fig. 2D show that the fluorescence intensity of the non-efflux group (as a positive control) is the strongest, proving the accumulation of rhodamine 6G in the cell; the untreated group only shows very low background fluorescence signal. The control group shows low fluorescence intensity, proving that rhodamine 6G is completely effluxed without the addition of inhibitors; and after treatment with 4 μg / mL CM-1, the intracellular fluorescence intensity is significantly enhanced; when the concentration of CM-1 is increased to 8 μg / mL, the fluorescence intensity is further increased, indicating that CM-1 has an inhibitory effect on the fungal efflux process, and the effect is dose-dependent. Figure 2 In Fig. 2B and Fig. 2E, the untreated group also only shows the background fluorescence level, the control group shows low fluorescence intensity, and after the addition of CM-1, the fluorescence peak is significantly right-shifted, indicating that the intracellular fluorescence substrate accumulation is increased, further proving that CM-1 can effectively inhibit the efflux effect.

[0061] Example 2

[0062] The experimental strains grown in the logarithmic phase were collected by centrifugation and washed with PBS buffer to remove the culture medium, and the cell density was determined by spectrophotometer, and then diluted to 1×10 3 The cell density was determined by spectrophotometer, and then diluted to 1×10 80 )。

[0063] Synergistic evaluation: The combination of antibacterial drugs can show "independent", "additive", "synergistic" and "antagonistic" effects in vitro or in animals. The results of combined drug sensitivity test are judged by fractional inhibitory concentration index.

[0064] Fractional inhibitory concentration index (FICI) is calculated as follows:

[0065] ΣFICI=FIC A +FIC B =C A / MIC A +C B / MIC B ;

[0066] Wherein, FICI is the fractional inhibitory concentration index, FIC A and FIC B respectively represent the MIC value of drug A and B combined with drug, MIC A and MIC B are the minimum inhibitory concentration of drug A and B alone, C A and C B are the concentrations of the two drugs combined to achieve the same efficacy. FICI>4 is antagonistic, FICI between 0.5 and 4 is additive or independent, and FICI≤0.5 is defined as synergistic.

[0067] The effect of CM-1 combined with azole drugs on antifungal activity is shown in Tables 1-8.

[0068] Table 1 CM-1 combined with fluconazole for antifungal activity

[0069]

[0070] In Table 1, MIC: minimum inhibitory concentration; MIC A : the minimum inhibitory concentration of CM-1 when the drug is used alone; C A : the minimum inhibitory concentration of CM-1 when the drugs are combined; MIC B : the minimum inhibitory concentration of fluconazole when the drug is used alone; C B : the minimum inhibitory concentration of fluconazole when the drugs are combined; FICI: fractional inhibitory concentration index; IN: combination index; SYN: synergistic effect.

[0071] Table 2 CM-1 combined with itraconazole for antifungal activity

[0072]

[0073] In Table 2, MIC: minimum inhibitory concentration; MIC A : the minimum inhibitory concentration of CM-1 when the drug is used alone; C A : the minimum inhibitory concentration of CM-1 when the drugs are combined; MIC B : the minimum inhibitory concentration of itraconazole when the drug is used alone; C B : the minimum inhibitory concentration of itraconazole when the drugs are combined; FICI: fractional inhibitory concentration index; IN: combination index; SYN: synergistic effect.A : Minimum inhibitory concentration of CM-1 alone; C A : Minimum inhibitory concentration of CM-1 in combination; MIC B : Minimum inhibitory concentration of itraconazole alone; C B : Minimum inhibitory concentration of itraconazole in combination; FICI: Fractional inhibitory concentration index; IN: Index of combination; SYN: Synergism.

[0074] Table 3 Antifungal effect of CM-1 in combination with voriconazole

[0075]

[0076] In Table 3, MIC: Minimum inhibitory concentration; MIC A : Minimum inhibitory concentration of CM-1 alone; C A : Minimum inhibitory concentration of CM-1 in combination; MIC B : Minimum inhibitory concentration of voriconazole alone; C B : Minimum inhibitory concentration of voriconazole in combination; FICI: Fractional inhibitory concentration index; IN: Index of combination; SYN: Synergism.

[0077] Table 4 Antifungal effect of CM-1 in combination with posaconazole

[0078]

[0079] In Table 4, MIC: Minimum inhibitory concentration; MIC A : Minimum inhibitory concentration of CM-1 alone; C A : Minimum inhibitory concentration of CM-1 in combination; MIC B : Minimum inhibitory concentration of posaconazole alone; C B : Minimum inhibitory concentration of posaconazole in combination; FICI: Fractional inhibitory concentration index; IN: Index of combination; SYN: Synergism.

[0080] Table 5 Antifungal effect of CM-1 in combination with clotrimazole

[0081]

[0082] In Table 5, MIC: Minimum inhibitory concentration; MIC A : Minimum inhibitory concentration of CM-1 alone; C A : Minimum inhibitory concentration of CM-1 in combination; MIC B : Minimum inhibitory concentration of clotrimazole alone; C BMIC: minimum inhibitory concentration; MIC: minimum inhibitory concentration; FICI: fractional inhibitory concentration index; IN: interaction index; SYN: synergy.

[0083] Table 6 Antifungal effect of CM-1 in combination with miconazole

[0084]

[0085] In Table 6, MIC: minimum inhibitory concentration; MIC: minimum inhibitory concentration; FICI: fractional inhibitory concentration index; IN: interaction index; SYN: synergy. A MIC of CM-1 when used alone; C A MIC of CM-1 when used in combination; MIC B MIC of miconazole when used alone; C B MIC of miconazole when used in combination; FICI: fractional inhibitory concentration index; IN: interaction index; SYN: synergy.

[0086] Table 7 Antifungal effect of CM-1 in combination with ketoconazole

[0087]

[0088] In Table 7, MIC: minimum inhibitory concentration; MIC: minimum inhibitory concentration; FICI: fractional inhibitory concentration index; IN: interaction index; SYN: synergy. A MIC of CM-1 when used alone; C A MIC of CM-1 when used in combination; MIC B MIC of ketoconazole when used alone; C B MIC of ketoconazole when used in combination; FICI: fractional inhibitory concentration index; IN: interaction index; SYN: synergy.

[0089] Table 8 Antifungal effect of CM-1 in combination with econazole

[0090]

[0091] In Table 8, MIC: minimum inhibitory concentration; MIC: minimum inhibitory concentration; FICI: fractional inhibitory concentration index; IN: interaction index; SYN: synergy. A MIC of CM-1 when used alone; C A MIC of CM-1 when used in combination; MIC B MIC of econazole when used alone; C B MIC of econazole when used in combination; FICI: fractional inhibitory concentration index; IN: interaction index; SYN: synergy.

[0092] As can be seen from the results in Tables 1-8, CM-1 itself has no antibacterial activity against Candida albicans, C. auris, C. glabrata, C. tropicalis, Trichophyton rubrum and T. indiana, but can reverse the drug resistance of Cdr1 and Mdr1 overexpressed drug-resistant fungi, and can synergistically inhibit the activity of C. albicans, C. auris, C. glabrata, C. tropicalis, T. rubrum and T. indiana in combination with azole drugs, effectively reducing the therapeutic dose of azole drugs, and having small drug toxicity.

[0093] Example 3

[0094] Cytotoxicity test of CM-1:

[0095] Cell lines: HEK293 human embryonic kidney cells, BEAS-2B human bronchial epithelial cells and HUVEC human umbilical artery epithelial cells.

[0096] CM-1 was dissolved in dimethyl sulfoxide (DMSO) to form a 10 mM stock solution.

[0097] Cells in the logarithmic growth phase were trypsinized, and the cell suspension was collected in a sterile centrifuge tube. The cell density was adjusted to 5x10 4 Cells / mL, inoculated in a 96-well plate and incubated overnight to allow the cells to adhere. The supernatant was replaced with fresh RPMI-1640 medium containing different concentrations of CM-1, and the 96-well plate was placed in a 37°C incubator containing 5% CO2 for 24 h. After incubation, the cell morphology was observed, and 10 μL of 5 mg / mL thiazolyl blue (MTT) solution was added to each well. After 4 h, the supernatant was discarded, 100 μL of DMSO was added to each well, and the absorbance at 490 nm was measured using a microplate reader.

[0098] The results are shown in Table 9, which show that CM-1 has good low cytotoxicity characteristics for HEK293, RAW264.7 and HUVEC.

[0099] Table 9 Results of cytotoxicity test

[0100]

[0101] Example 4

[0102] In vivo antibacterial verification:

[0103] In single and combination drug experiments, 6-8 weeks old male BALB / c mice were randomly divided into four groups (10 mice per group): CM-1 single drug treatment group, fluconazole single drug treatment group, CM-1 combined with fluconazole treatment group and normal saline control group. The overnight activated white Candida GU5 or white Candida G5 yeast phase cells were adjusted to 1×10 7 cells / mL with sterile normal saline, and 100 μL of the bacterial suspension was injected into each mouse via the tail vein. After 24 h of infection, the drug administration regimen was started: the CM-1 single drug treatment group was injected with 20 mg / kg CM-1 intraperitoneally, the fluconazole single drug treatment group was injected with 5 mg / kg fluconazole, the CM-1 combined with fluconazole treatment group was injected with 20 mg / kg CM-1 and 5 mg / kg fluconazole simultaneously, and the control group was injected with the same volume of sterile normal saline. The drug was administered once a day for 3 consecutive days. The survival rate was recorded daily during the treatment period, and the bilateral kidneys were collected after humane euthanasia on the 4th day: the left kidney was fixed with 4% paraformaldehyde for histopathological analysis, and the fixed tissue was embedded in paraffin for sectioning, then PAS and H&E staining were used for histomorphological observation and fungal infiltration evaluation; the right kidney was used for real-time detection of kidney fungal load.

[0104] The results are shown in Figure 3 and Figure 4 It can be seen that the survival curves of the CM-1 or fluconazole treatment groups alone have no significant difference from the PBS group. However, the survival rate of the CM-1 combined with fluconazole treatment group is significantly higher than that of the single drug treatment group. Kidney fungal load analysis shows that the fungal pathogenicity of the CM-1 combined with fluconazole treatment group is significantly reduced. Histopathological examination shows that there are few Candida albicans in the kidney of the CM-1 combined with fluconazole treatment group, and the inflammatory cell infiltration of the kidney tissue is significantly improved. These findings indicate that the CM-1 combined with fluconazole treatment regimen has important clinical application potential as a new anti-drug resistant fungal infection strategy.

[0105] Among them, in the Candida albicans G5 group, the control group had a bacterial load of 2.84×10 8 CFU / g, the CM-1 single drug treatment group had a bacterial load of 4.66×10 8 CFU / g, the fluconazole single drug treatment group had a load of 1.27×10 8 CFU / g, and the CM-1 combined with fluconazole treatment group had a bacterial load of 4.75×10 7 CFU / g;

[0106] In the Candida albicans GU5 group, the control group had a bacterial load of 3.40×10 8 CFU / g, the CM-1 single drug treatment group had a bacterial load of 3.83×10 8 CFU / g, the fluconazole single drug treatment group had a load of 4.09×10 8CFU / g, CM-1 combined with fluconazole treatment group was 1.60 x 10 7 CFU / g.

[0107] The bacterial load of each group was taken as the average value.

[0108] Example 5

[0109] Compared with Example 4, the drug ratio of CM-1 and fluconazole was adjusted, and the other experimental steps were the same as Example 4. Specifically, the CM-1 single drug treatment group was injected with 20 mg / kg CM-1, the fluconazole single drug treatment group was injected with 3 mg / kg fluconazole, the CM-1 combined with fluconazole treatment group was injected with 20 mg / kg CM-1 and 3 mg / kg fluconazole at the same time, and the control group was injected with the same volume of sterile normal saline.

[0110] The results are shown in Table 10. As can be seen from the table, the kidney fungal load analysis showed that the fungal pathogenicity of the CM-1 combined with fluconazole treatment group was significantly reduced, but compared with the simultaneous injection of 20 mg / kg CM-1 and 5 mg / kg fluconazole in Example 4, the kidney fungal load of Candida albicans GU5 infection increased. These findings indicate that the CM-1 combined with fluconazole treatment regimen is related to the dosage of the drugs used.

[0111] Table 10 Verification of the effect of CM-1 combined with fluconazole on a mouse systemic infection model

[0112]

[0113] Example 6

[0114] Compared with Example 4, the drug ratio of CM-1 and fluconazole was adjusted, and the other experimental steps were the same as Example 4. Specifically, the CM-1 single drug treatment group was injected with 10 mg / kg CM-1, the fluconazole single drug treatment group was injected with 7 mg / kg fluconazole, the CM-1 combined with fluconazole treatment group was injected with 10 mg / kg CM-1 and 7 mg / kg fluconazole at the same time, and the control group was injected with the same volume of sterile normal saline.

[0115] The results are shown in Table 11. As can be seen from the table, the kidney fungal load analysis showed that the fungal pathogenicity of the CM-1 combined with fluconazole treatment group was significantly reduced, but compared with the simultaneous injection of 20 mg / kg CM-1 and 5 mg / kg fluconazole in Example 4, the kidney fungal load increased. These findings indicate that the CM-1 combined with fluconazole treatment regimen is related to the dosage of the drugs used.

[0116] Table 11 Verification of the effect of CM-1 combined with fluconazole on a mouse systemic infection model

[0117]

[0118] Example 7

[0119] Compared with Example 4, the drug ratio of CM-1 and fluconazole was adjusted, and other experimental procedures were the same as Example 4. Specifically, the CM-1 monotherapy group was 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 injected with 6 mg / kg CM-1 and 6 mg / kg fluconazole at the same time, and the control group was injected with the same volume of sterile normal saline.

[0120] The results are shown in Table 12. As can be seen from the table, the kidney fungal load analysis showed that the fungal pathogenicity of the CM-1 combined with fluconazole treatment group was significantly reduced, but compared with the simultaneous injection of 20 mg / kg CM-1 and 5 mg / kg fluconazole in Example 4, the kidney fungal load increased. These findings indicate that the CM-1 combined with fluconazole treatment regimen is related to the dosage of the drugs used.

[0121] Table 12 Verification of the effect of CM-1 combined with fluconazole in a mouse systemic infection model

[0122]

[0123] Example 8

[0124] Compared with Example 4, the drug ratio of CM-1 and fluconazole was adjusted, and other experimental procedures were the same as Example 4. Specifically, the CM-1 monotherapy group was 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 injected with 60 mg / kg CM-1 and 3 mg / kg fluconazole at the same time, and the control group was injected with the same volume of sterile normal saline.

[0125] The results are shown in Table 13. As can be seen from the table, the kidney fungal load analysis showed that the fungal pathogenicity of the CM-1 combined with fluconazole treatment group was significantly reduced, but compared with the simultaneous injection of 20 mg / kg CM-1 and 5 mg / kg fluconazole in Example 4, the kidney fungal load increased. These findings indicate that the CM-1 combined with fluconazole treatment regimen is related to the dosage of the drugs used.

[0126] Table 13 Verification of the effect of CM-1 combined with fluconazole in a mouse systemic infection model

[0127]

[0128] Example 9

[0129] Compared with Example 4, the species of fungi were adjusted to C. auris CBS12774, C. glabrata CG2, C. tropicalis CT2, and other experimental procedures were the same as Example 4.

[0130] The results are shown in Table 14. From the table, it can be seen that the fungal load of the CM-1 alone group in the mouse model infected with C. auris CBS12774, C. glabrata CG2, C. tropicalis CT2 was not significantly different from the control group. However, the kidney fungal load analysis of the combination therapy group showed that the pathogenicity of fungi was significantly reduced in the combination therapy group. These findings indicate that the combination therapy of CM-1 and fluconazole has important application potential for the treatment of infections with multiple drug-resistant bacteria.

[0131] Table 14. Verification of the effect of the combination of CM-1 and fluconazole in a mouse systemic infection model

[0132]

[0133] Example 10

[0134] Verification of the topical combination of CM-1 and fluconazole for Trichophyton rubrum and T. indiana in a mouse skin infection model:

[0135] Male BALB / c mice weighing 20-22 g were randomly housed in individual cages with four mice per cage and acclimated 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 perforation was performed on the back skin using a biopsy punch with a diameter of 0.8 cm. Suspensions of T. rubrum and T. indiana (1 x 10 8 CFU / mL, 50 μL per mouse) were inoculated into the circular wound until the skin appeared wet but with no excess liquid; 24 h after inoculation, about 30 mg of different ointments was applied to the skin infection site, respectively, an ointment containing 2% (mass fraction) CM-1, an ointment containing 1% (mass fraction) fluconazole, and an ointment containing both 2% (mass fraction) CM-1 and 1% (mass fraction) fluconazole, and covered with a circular paraffin film; at the same time, an ointment containing only the base was used as a negative control, and all ointments were applied at 12 h intervals, for a total of 3 times; on day 8 after infection, the fungal count of the wound specimen was recorded.

[0136] Results are shown in Table 15. As can be seen from the table, the fungal load in the CM-1 alone group was not significantly different from the control group in both the T. rubrum and T. mentagrophytes mouse model; however, the fungal pathogenicity was significantly reduced in the combination therapy group. These findings suggest that the combination therapy of CM-1 and fluconazole has important application potential for the treatment of infections of multiple drug-resistant bacteria.

[0137] Table 15. Verification of the external use combination effect of CM-1 and fluconazole on T. rubrum and T. mentagrophytes in a mouse skin infection model

[0138]

[0139] Example 11

[0140] Verification of the external use combination of CM-1 and clotrimazole in a mouse skin infection model:

[0141] Compared with Example 10, the species of fungi were modified to C. albicans GU5, C. auris CBS12774, C. glabrata CG2, C. tropicalis CT2, T. rubrum and T. mentagrophytes, respectively; after successfully creating a mouse skin infection model infected with different fungi, about 30 mg of different ointments were applied to the site of skin infection, respectively, 2% (mass fraction) CM-1 ointment, 1% (mass fraction) clotrimazole ointment and 2% (mass fraction) CM-1 and 1% (mass fraction) clotrimazole ointment at the same time, and covered with a round paraffin film; the other experimental processes were the same as those of Example 10.

[0142] Results are shown in Table 16.

[0143] Table 16. Verification of the external use combination effect of CM-1 and clotrimazole in a mouse skin infection model

[0144]

[0145] Example 12

[0146] Verification of the external use combination effect of CM-1 and miconazole in a mouse skin infection model:

[0147] Compared with Example 11, the azole drug in Example 11 was modified to miconazole, and the other methods were the same as those of Example 11.

[0148] Results are shown in Table 17.

[0149] Table 17. Verification of the external use combination effect of CM-1 and miconazole in a mouse skin infection model

[0150]

[0151] Example 13

[0152] The mouse skin infection model was used to verify the external use of CM-1 and ketoconazole:

[0153] 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.

[0154] The results are shown in Table 18.

[0155] Table 18. The mouse skin infection model was used to verify the external use of CM-1 and ketoconazole

[0156]

[0157] Example 14

[0158] The mouse skin infection model was used to verify the external use of CM-1 and econazole:

[0159] Compared with Example 11, the azole drug in Example 11 was changed to econazole, and the other methods were the same as those in Example 11.

[0160] The results are shown in Table 19.

[0161] Table 19. The mouse skin infection model was used to verify the external use of CM-1 and econazole

[0162]

[0163] The results in Tables 16-19 show that CM-1 combined with clotrimazole, miconazole, ketoconazole and econazole, respectively, has a significant synergistic effect in treating mouse skin fungal infections, can quickly eliminate pathogens, reduce tissue inflammation and promote skin healing. The present application provides a new way to overcome azole resistance, not only reveals the great potential of CM-1 as an antifungal synergist, but also lays a solid theoretical and experimental foundation for developing more efficient and safer combined therapy for superficial fungal infections, and has important clinical translation value.

[0164] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. The application of the compound shown in formula (Ⅰ) in the preparation of drugs that enhance the antifungal effect of azole drugs; Equation (I); The azole drugs are one or more of clotrimazole, miconazole, econazole, fluconazole, ketoconazole, voriconazole, posaconazole and itraconazole; The fungus is one or more of the following: Candida albicans, Candida tropicalis, Candida glabrata, Candida auris, Trichophyton rubrum, and Trichophyton indicum. The mass ratio of the compound shown in formula (Ⅰ) to the azole drug is (1~200):

1.

2. The application of the compound shown in formula (Ⅰ) in the preparation of drugs that reduce fungal resistance to azole drugs; Equation (I); The azole drugs are one or more of clotrimazole, miconazole, econazole, fluconazole, ketoconazole, voriconazole, posaconazole and itraconazole; The fungus is one or more of the following: Candida albicans, Candida tropicalis, Candida glabrata, Candida auris, Trichophyton rubrum, and Trichophyton indicum. The mass ratio of the compound shown in formula (Ⅰ) to the azole drug is (1~200):

1.

3. The application of the compound shown in formula (Ⅰ) in combination with azole drugs in the preparation of antifungal drugs; Equation (I); The azole drugs are one or more of clotrimazole, miconazole, econazole, fluconazole, ketoconazole, voriconazole, posaconazole and itraconazole; The fungal package is one or more of the following: Candida albicans, Candida tropicalis, Candida glabrata, Candida auris, Trichophyton rubrum, and Trichophyton indicum. The mass ratio of the compound shown in formula (Ⅰ) to the azole drug is (1~200):

1.

4. An antifungal drug, characterized in that, Includes compounds shown in formula (Ⅰ) and azole drugs; Equation (I); The azole drugs are one or more of clotrimazole, miconazole, econazole, fluconazole, ketoconazole, voriconazole, posaconazole and itraconazole; The mass ratio of the compound shown in formula (Ⅰ) to the azole drug is (1~200):1; The fungus is one or more of the following: Candida albicans, Candida tropicalis, Candida glabrata, Candida auris, Trichophyton rubrum, and Trichophyton indicum.

5. A pharmaceutical composition, characterized in that, Including the antifungal drug as described in claim 4.

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