Use of nimesulide in combination with an azole or allylamine antifungal agent for the preparation of an antifungal agent

Abstract

The application discloses application of nimesulide or a pharmaceutical salt thereof combined with an azole antifungal drug or a propenylamine antifungal drug in preparation of an antifungal drug. The application proves through in-vivo and in-vitro experiments that nimesulide combined with fluconazole shows clear synergistic antifungal effect. Nimesulide combined with fluconazole can improve the survival rate of animals infected with fungi, and has low toxicity to wax moth larvae and mammal cells.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology, specifically, it relates to the application of nimesulide in combination with azole antifungal drugs or allylamine antifungal drugs in the preparation of antifungal drugs. Background Technology

[0002] In recent decades, the incidence of fungal infections has been steadily increasing. With the widespread use of hematopoietic stem cell transplantation, solid organ transplantation, chemotherapy for tumors, broad-spectrum antibiotics, glucocorticoids, and immunosuppressants, the increase in sepsis patients, and the development of interventional diagnostic and treatment methods such as intravenous nutrition, the population with weakened immune function and dysbiosis has been expanding, leading to a dramatic increase in the incidence and mortality of invasive fungal infections (IFIs). Invasive candidiasis (IC) constitutes a major category of IFIs, with a mortality rate roughly estimated to be over 40%, among which Candida albicans (C. albicans) is the most prevalent. The main infecting strain is *Candida*. Currently, the types of drugs available clinically for treating invasive fungal infections are very limited, mainly falling into five categories: polyenes, such as amphotericin B and its liposomal preparations; azoles, such as fluconazole (FLC), itraconazole, and voriconazole; allylamines, such as terbinafine; echinocandins, such as caspofungin; and pyrimidines, such as 5-fluorocytosine. However, with the widespread and frequent use of antifungal drugs, pathogenic fungi have long since evolved drug resistance, and the resistance situation is becoming increasingly severe. Drug resistance seriously hinders the effective implementation of clinical antifungal treatment. Furthermore, studies on bloodstream infections of *Candida* have found that a considerable number of patients, despite being infected with so-called sensitive fungi to which drugs have low minimum inhibitory concentrations (MICs), still cannot be effectively eradicated by existing antifungal drugs. This is attributed to fungal resistance, a phenomenon that is increasingly attracting attention in the field of fungal therapy. Given the high morbidity and mortality rates of fungal infections, the inadequacy of existing antifungal drugs, and the continuous emergence of drug-resistant and resistant strains, there is an urgent need to discover new antifungal strategies, drug targets, and new drugs.

[0003] Developing novel antifungal drugs with entirely new structural types is important, but it involves long research cycles, high funding requirements, and the risk of clinical trial failure. Using existing drugs as potentiators for antifungal drugs has the advantages of low development costs, high efficiency, and broad clinical application prospects. Studies have shown that nimesulide (NIM) is effective against Trichophyton mentagrophytes (…). It has antibacterial activity, with a MIC value of 2 μg / mL, and can also inhibit Cryptococcus neoformans (Cryptococcus neoformans). ) and Cryptococcus grease ( The MIC for nimesulide was 62 μg / mL. Other studies have shown that nimesulide at a concentration of 1 mM has a mild (5-15%) inhibitory effect on Candida albicans biofilm formation. However, there are no reports on nimesulide as a potentiator for existing antifungal drugs. Summary of the Invention

[0004] The purpose of this invention is to provide the application of nimesulide (NIM) in combination with azole antifungal drugs or allylamine antifungal drugs in the preparation of antifungal drugs, so as to better solve the technical problems of treating fungal infectious diseases and fungal resistance and tolerance to antifungal drugs.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides the use of nimesulide or its pharmaceutical salts in combination with azole antifungal drugs or allylamine antifungal drugs in the preparation of antifungal drugs.

[0007] The structure of nimesulide (Caiazzo E, Ialenti A, Cicala C. The relatively selective cyclooxygenase-2 inhibitor nimesulide; Eur J Pharmacol. 2019;848:105-111.) is shown below:

[0008]

[0009] The azole antifungal drugs are selected from fluconazole (FLC), itraconazole, and voriconazole.

[0010] The allylamine antifungal drug is selected from terbinafine.

[0011] The medicinal salts referred to are acid addition salts formed by nimesulide with the following acids: hydrochloric acid, sulfuric acid, nitric acid, citric acid, maleic acid, etc.

[0012] The fungus is selected from Candida albicans and Candida tropicalis, specifically: Candida albicans ( The strain is selected from SC5314, 904, 901, 632, 103, 100, 953, 9821, 9296, Y0109, and 7654; the tropical Candida ( (Selected from 8915 and 409.)

[0013] This invention has found that, for fluconazole-resistant Candida albicans, the combined use of nimesulide and azole antifungal drugs can reduce the effective concentration of the drugs, decreasing the effective concentration of azole antifungal drugs from 8-32 μg / mL to 0.5-2 μg / mL.

[0014] The preferred combination regimen is: nimesulide concentration of 1~32 μg / mL + fluconazole concentration of 0.5~16 μg / mL.

[0015] The preferred combination regimen is: nimesulide concentration of 2~4 μg / mL + itraconazole concentration of 0.25~1 μg / mL.

[0016] The preferred combination regimen is: nimesulide concentration of 2~8 μg / mL + voriconazole concentration of 0.25~0.5 μg / mL.

[0017] The preferred combination of these two treatments is: nimesulide at a concentration of 32-128 μg / mL + terbinafine at a concentration of 4-128 μg / mL.

[0018] For fluconazole-resistant Candida tropicalis, the preferred combination therapy is: nimesulide at a concentration of 2-32 μg / mL + fluconazole at a concentration of 16-64 μg / mL.

[0019] In a second aspect, the present invention provides a pharmaceutical preparation made from nimesulide or its pharmaceutical salts combined with azole antifungal drugs or allylamine antifungal drugs.

[0020] The dosage form of the pharmaceutical preparation is selected from liquid preparations, tablets, or capsules.

[0021] The drug preparation can be administered orally, intravenously, intraperitoneally, or via enema.

[0022] A third aspect of the present invention provides the use of the nimesulide or its pharmaceutical salt in the preparation of synergists for azole antifungal drugs or allylamine antifungal drugs.

[0023] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0024] This invention reveals that the clinically commonly used nonsteroidal anti-inflammatory drug (NIM) exhibits a clear synergistic antifungal effect when used in combination with azole antifungal drugs, and that NIM shows a synergistic or additive effect when used in combination with allylamine antifungal drugs. Animal fungal infection experiments show that the combination of NIM and FLC improves the survival rate of fungal-infected animals, further demonstrating that NIM has a good effect in enhancing the antifungal efficacy of FLC.

[0025] This invention has found that when NIM and FLC are used together to combat resistant fungi, a low concentration (2 μg / mL) of NIM combined with FLC can inhibit the growth of FLC-resistant fungi.

[0026] This invention clarifies that the combined use of NIM and azole drugs exhibits synergistic antifungal activity both in vivo and in vitro. Since infections are often accompanied by fever, anti-infective drugs are frequently used in combination with antipyretics, analgesics, and anti-inflammatory drugs. NIM is an FDA-approved antipyretic, analgesic, and anti-inflammatory drug that exerts its antipyretic, analgesic, and anti-inflammatory effects by selectively inhibiting COX2. Its combination with azole drugs demonstrates a synergistic antifungal effect. This method is characterized by low development costs, high efficiency, and broad clinical application prospects.

[0027] Mechanism studies of this invention have revealed that NIM acts as an antifungal drug synergist not by inhibiting cyclooxygenase 2 (COX2), not by affecting the fungal ergosterol synthesis pathway, and not by inhibiting the function of the fungal drug efflux pump, but based on other novel mechanisms of action.

[0028] Both cell and animal experiments consistently showed that the combination of the two drugs did not exhibit toxicity even at concentrations ten to dozens of times higher than the effective concentration, indicating that the combination of NIM and FLC has a high safety profile. Attached Figure Description

[0029] Figure 1 This is a schematic diagram showing the results of the inhibition zone determination of NIM combined with azole drugs against drug-resistant fungi in Example 3.

[0030] Figure 2 This is a schematic diagram showing the results of the determination of the inhibition zone of resistant fungi when NIM and FLC are used together in Example 4.

[0031] Figure 3 This is a schematic diagram showing the time-growth curve results of the combined use of NIM and FLC to combat drug-resistant fungi in Example 5.

[0032] Figure 4 This is a schematic diagram showing the results of Example 6, in which the combined use of NIM and FLC effectively improved the survival rate of wax moth larvae infected with drug-resistant Candida albicans.

[0033] Figure 5 This is a schematic diagram of the cytotoxicity results of the combined use of NIM and FLC in Example 7.

[0034] Figure 6 This is a schematic diagram of the toxicity results of NIM and FLC used in combination for wax moth larvae in Example 8.

[0035] Figure 7 This is a schematic diagram showing the results of the inhibition zone determination of drug-resistant fungi when nonsteroidal anti-inflammatory drugs are used in combination with FLC in Example 9.

[0036] Figure 8This is a schematic diagram showing the effect of PGE2 on the combined use of NIM and FLC against drug-resistant fungi in Example 10.

[0037] Figure 9 This is a schematic diagram showing the effect of AA on the combined use of NIM and FLC against drug-resistant fungi in Example 10.

[0038] Figure 10 This is a schematic diagram showing the comparison results of human COX2 homologous genes or proteins in Candida albicans in Example 11.

[0039] Figure 11 This is a schematic diagram showing the effect of thin-layer chromatography on the ergosterol content of NIM and FLC in Example 12.

[0040] Figure 12 This is a schematic diagram showing the results of the experiment in Example 13 where we explored the drug concentration and action time for GC-MS detection of sterol components.

[0041] Figure 13 This is a gas chromatogram of various sterol components of Candida albicans after drug treatment, as detected by GC-MS in Example 13.

[0042] Figure 14 This is a schematic diagram showing the effect of GC-MS detection of the combined use of NIM and FLC on the proportion of various sterol components in the total sterols of Candida albicans in Example 13.

[0043] Figure 15 This is a schematic diagram showing the results of the determination of the effect of NIM on the drug efflux pump of Candida albicans in Example 14. Detailed Implementation

[0044] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0045] This invention reveals that the compound NIM, when used in combination with azole drugs, exhibits good in vitro anti-drug resistance and antifungal activity, and the combination of NIM and azole drugs demonstrates high biocompatibility. The effect of NIM as an antifungal drug synergist is not achieved by blocking the arachidonic acid pathway, affecting the ergosterol synthesis pathway in Candida albicans, or inhibiting the drug efflux pump in Candida albicans.

[0046] In this invention, the fungi used can be obtained or cultured by conventional methods, or can be obtained commercially, such as isolated from somatic cells or tissues of patients with the corresponding disease, or purchased from any institution or company that provides commercially available standard strains, such as ATCC. It should be understood that the strain samples used in the embodiments of this invention are merely exemplary examples, and any fungi resistant to or tolerant of FLC can be used to test the antifungal activity of the combination therapy of this invention, without being limited to the scope of the embodiments.

[0047] Example 1

[0048] Determination of the partial inhibitory concentration index (FICI) of NIM combined with FLC

[0049] Materials and methods:

[0050] 1. Medicinal Materials: NIM was purchased from Macklin Chemical Co., Ltd., FLC from Shanghai Aladdin Biochemical Technology Co., Ltd., and dimethyl sulfoxide (DMSO) from Sinopharm Chemical Reagent Co., Ltd. NIM and FLC were prepared into 10 mg / mL solutions with DMSO. The test drugs were stored at -20°C. Before the experiment, the drug stock solutions were removed and thawed at room temperature, thoroughly mixed, and then used for subsequent experiments.

[0051] 2. Strains: The tested fungi include Candida albicans ( ), Tropical Candida ( Ten strains were collected, including *Candida albicans* 901 and 953, which were provided by the Department of Pharmaceutical Chemistry, Department of Pharmacy, Naval Medical University, and the remaining strains by the Department of Dermatology, Shanghai Changzheng Hospital. The strains were mixed with 80% glycerol at a 1:1 volume ratio and stored at -80°C. For activation, a small amount of the frozen strain was taken out and added to a shaker tube containing 1 mL of YPD liquid medium. The culture was incubated at 30°C and 200 rpm for 24 h with shaking. 10 μL of this culture was then added to a shaker tube containing 1 mL of YPD liquid medium and cultured under the same conditions for another 16 h to bring the strain to the logarithmic growth phase. This culture was then used for subsequent experiments.

[0052] 3. Culture medium

[0053] RPMI 1640 medium: RPMI 1640 (Gibco BRL) 10.0 g, NaHCO3 2.0 g, morpholinepropanesulfonic acid (MOPS) (Sigma) 34.5 g (0.165 M), dissolved in 900 mL of triple-distilled water, add an appropriate amount of NaOH to adjust the pH of the solution to 7.0 (25℃), bring the volume to 1000 mL, filter to sterilize, and store at 4℃.

[0054] YPD liquid culture medium: 10.0 g yeast extract (BD Biosciences, USA), 20.0 g peptone (BD Biosciences, USA), 20.0 g glucose (Shanghai Sangon Biotech Co., Ltd.), dissolved in 900 mL triple-distilled water, brought to a final volume of 1000 mL, autoclaved and stored at 4°C.

[0055] 4. Instruments: Biosafety cabinet (BSC-1004II A2, Suzhou Antai Air Technology Co., Ltd.); biological microscope (LW100T, Beijing Cewei Optoelectronic Technology Co., Ltd.); constant temperature incubator (MJ-150-I, Shanghai Yiheng Scientific Instrument Co., Ltd.); shaking incubator (HZ-2111K-B, Taicang Experimental Equipment Factory, Jiangsu Province); vortex apparatus (Thermo Vortex Maxi Mix II); mini centrifuge (Hitachi CT15RE); multi-functional microplate reader (BioTek Synergy H1).

[0056] 5. Determination of FICI in combination with NIM and FLC using the checkerboard micro-liquid dilution method

[0057] The test strain was inoculated into YPD liquid medium and cultured at 30°C for 16 h. The activated bacterial suspension was transferred to a 1.5 mL centrifuge tube, centrifuged at 3000 g for 1 min, the medium was discarded, and the cells were washed twice with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1 mL PBS. After dilution, 10 μL was transferred to a hemocytometer for counting. The bacterial concentration was adjusted to 1 × 10⁻⁶ cells / mL using RPMI 1640 medium. 3Cells / mL. After mixing the prepared bacterial suspension, transfer it to a 12-well plate. Add 2.8 mL to the first well, and 1.4 mL to each of wells 2-7. Prepare stock solutions of NIM and FLC using DMSO. Add the NIM stock solution to the first well to achieve a final concentration of 64-128 μg / mL. Perform 50 / 20 dilutions sequentially to achieve final concentrations of 64-1 μg / mL or 256-4 μg / mL in wells 1-7, respectively. Transfer the prepared NIM-containing bacterial suspension to rows A and B of a 96-well plate according to drug concentration from highest to lowest: 200 μL / well in the first column, and 100 μL / well in columns 2-10. Add blank bacterial suspension without NIM to wells H1-H11 in row H: 200 μL / well for H1, and 100 μL / well for H2-H11. Add 100 μL / well of untreated blank bacterial suspension to wells A11-H11 in column 11 as a growth control. The prepared FLC was added to wells A1-H1 in the first column to a final concentration of 64 μg / mL. Each column was then halved from left to right to achieve final FLC concentrations of 64–0.25 μg / mL in columns 1–9. 100 μL of RPMI 1640 medium was added to each well in column 12 of the 96-well plate as a blank control. The 96-well plate was incubated at 30°C for 48 h, and its OD value was measured. 630 The OD of the growth control group was used as an example. 630 The value is 100%. Calculate the antibacterial rate (%) of the compound at different drug concentrations for each well, and obtain the MIC when NIM and FLC are used alone or in combination. 80 Then, the synergistic index FICI was calculated. The experimental results are shown in Table 1, which shows the determination results of the partial inhibitory concentration index (FICI) of NIM and FLC against various pathogenic fungi.

[0058] Table 1

[0059]

[0060] Table 1 shows that NIM and FLC have a synergistic antifungal effect when used together. The fractional inhibitory concentration index (FICI) is the main parameter for evaluating the interaction between the two drugs in combination. FICI is calculated as the sum of the ratios of the minimum inhibitory concentration (MIC) when the two drugs are used together to the MIC when used alone, i.e., FICI = FICA + FICB = (MICAB / MICA) + (MICBA / MICB), where MICAB and MICBA are the minimum inhibitory concentrations of drug A and drug B respectively when used together, and MICA and MICB are the minimum inhibitory concentrations of drug A and drug B when used alone. This experiment used internationally recognized standards to evaluate the combined drug effect: FICI ≤ 0.5, synergistic effect, and the smaller the FICI value, the stronger the synergistic effect; 0.5 < FICI ≤ 1, additive effect; 1 < FICI ≤ 4, no effect; FICI > 4, antagonistic effect. The results of the checkerboard micro-liquid dilution method showed that the FICI of NIM and FLC combined against Candida albicans and Candida tropicalis was less than 0.5, indicating that the combination of the two drugs had a clear synergistic antifungal effect.

[0061] Example 2

[0062] Determination of the partial inhibitory concentration index (FICI) of NIM in combination with existing antifungal drugs

[0063] Materials and methods

[0064] 1. Drugs: NIM, itraconazole, voriconazole, and terbinafine were purchased from Macklin Pharmaceuticals; FLC was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; dimethyl sulfoxide (DMSO) was purchased from Sinopharm Chemical Reagent Co., Ltd. NIM, FLC, itraconazole, voriconazole, and terbinafine were prepared into 10 mg / mL solutions using DMSO. The test drugs were stored at -20°C. Before the experiment, the drug stock solutions were thawed at room temperature, thoroughly mixed, and then used for subsequent experiments.

[0065] 2. Strains: Drug-resistant Candida albicans 904 and 100 were provided by the Department of Dermatology, Shanghai Changzheng Hospital; drug-resistant Candida albicans 901 was provided by the Department of Pharmaceutical Chemistry, Department of Pharmacy, Naval Medical University. The methods for cryopreservation and activation of the strains were the same as in Example 1.

[0066] 3. Culture medium: Same as in Example 1.

[0067] 4. Instruments: Same as in Example 1.

[0068] 5. Determination of FICI of NIM in combination with existing antifungal drugs using checkerboard micro-liquid dilution method

[0069] The experimental method was the same as in Example 1. The experimental results are shown in Tables 2-1 and 2-2. Table 2-1 (MIC) 50 ), 2-2 (MIC) 80 The result is the partial inhibitory concentration index (FICI) of NIM combined with antifungal drugs.

[0070] Table 2-1

[0071]

[0072] Table 2-2

[0073]

[0074] Table 2 shows that the FICI values ​​of NIM combined with azole drugs FLC, itraconazole, and voriconazole against drug-resistant Candida albicans 904 and 901 were all less than 0.5, indicating that NIM and azole drugs have a synergistic effect against drug-resistant Candida albicans. The FICI values ​​of NIM combined with terbinafine against drug-resistant Candida albicans 904, 901, and 100 were equal to 0.5, or greater than 0.5 and less than 1, indicating that NIM combined with terbinafine has a synergistic or additive effect against drug-resistant Candida albicans.

[0075] According to the results in Tables 1, 2-1, and 2-2, for FLC-resistant Candida albicans, the combined use of NIM and azole antifungal drugs can reduce the effective concentration of the drugs, lowering it from 8–32 μg / mL to 0.5–2 μg / mL. Preferred combined use regimens are: NIM concentration 1–32 μg / mL + FLC concentration 0.5–16 μg / mL; NIM concentration 2–4 μg / mL + itraconazole concentration 0.25–1 μg / mL; NIM concentration 2–8 μg / mL + voriconazole concentration 0.25–0.5 μg / mL; NIM concentration 32–128 μg / mL + terbinafine concentration 4–128 μg / mL. For FLC-resistant Candida tropicalis, the preferred combination regimen is: NIM concentration of 2~32 μg / mL + FLC concentration of 16~64 μg / mL.

[0076] Example 3

[0077] Determination of inhibition zone of NIM in combination with existing antifungal drugs against drug-resistant Candida albicans

[0078] Materials and methods

[0079] 1. Medicinal materials: Same as in Examples 1 and 2.

[0080] 2. Strains: FLC-resistant Candida albicans 904 was provided by the Department of Dermatology, Shanghai Changzheng Hospital. The cryopreservation and activation methods for the strain were the same as in Example 1.

[0081] 3. Culture medium

[0082] YPD liquid medium: The preparation method is the same as in Example 1. YPD solid medium: Add 20.0 g of agar (BD Company, USA) to the YPD liquid medium, autoclave, and add filtered sterilized NIM solution when the medium is no longer hot to the touch, so that the final concentration of NIM in the medium is 8, 16, and 32 μg / mL, respectively. Then pour the medium into 90 mm Petri dishes and let it air dry for later use.

[0083] 4. Instruments: Biosafety cabinet (BSC-1004II A2, Suzhou Antai Air Technology Co., Ltd.); biological microscope (LW100T, Beijing Cewei Optoelectronic Technology Co., Ltd.); constant temperature incubator (MJ-150-I, Shanghai Yiheng Scientific Instrument Co., Ltd.); shaking incubator (HZ-2111K-B, Jiangsu Taicang Experimental Equipment Factory); vortex apparatus (Thermo Vortex Maximix II); mini centrifuge (Hitachi CT15RE).

[0084] 5. Paper diffusion experiment

[0085] Candida albicans was inoculated into YPD liquid medium and cultured at 30°C for 16 h. The activated bacterial suspension was transferred to a 1.5 mL centrifuge tube, centrifuged at 3000 g for 1 min, the medium was discarded, and the cells were washed twice with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1 mL PBS. After dilution, 10 μL was transferred to a hemocytometer for counting. The bacterial concentration was adjusted to 1 × 10⁻⁶ cells / mL with sterile PBS. 6Cells / mL. 100 μL of diluted bacterial suspension was poured onto agar plates containing different concentrations (8, 16, 32 μg / mL) of NIM, and sterile glass beads were added to spread the suspension evenly. Sterile filter paper discs (6 mm in diameter) were placed on solid culture media, with an equal volume (5 μL) of different concentrations (0.156–5 mg / mL) of FLC added to the center of each disc. The agar plates were inverted and incubated at 30°C. The formation of inhibition zones was observed at 24 h and 48 h, and the diameter of the inhibition zones was measured and photographed. To verify the dose-response relationship of NIM and FLC synergistically against drug-resistant fungi, a fixed amount (6.25 μg) of FLC was added to filter paper discs in agar plates, followed by different amounts (0–100 μg) of NIM for disc diffusion experiments. To investigate the antifungal effect of NIM in combination with other antifungal drugs, equal volumes of azole antifungal drugs (itraconazole and voriconazole) at different concentrations were added to filter paper discs on agar plates containing 16 μg / mL NIM. The experimental results are shown below. Figure 1 As shown, Figure 1 This is a schematic diagram showing the results of the inhibition zone determination of NIM combined with azole drugs against drug-resistant fungi in Example 3.

[0086] The results showed that: (1) Compared with the control agar plates without NIM, a clear inhibition zone was formed around the FLC-loaded filter paper on the NIM-loaded plates. No fungi grew inside the inhibition zone, and the inhibition zone showed a trend of increasing significantly with the increase of FLC loading. Figure 1 As shown in Figure A), it indicates that the combination of NIM and FLC has a synergistic effect against drug-resistant fungi, and this synergistic effect may be concentration-dependent with respect to FLC; (2) Compared with NIM or FLC alone, a significant inhibition zone was generated around the filter paper when quantitative FLC was used in combination with different amounts of NIM. Figure 1 (See Figure B); (3) Compared with the control agar plate without NIM, a clear inhibition zone was formed around the filter paper plate loaded with itraconazole or voriconazole on the plate containing NIM (16 μg / mL). No fungi grew inside the inhibition zone, and the inhibition zone showed a trend of increasing with the increase of itraconazole and voriconazole loading. Figure 1 (As shown in C). The above results indicate that NIM, when used in combination with azole drugs, has a synergistic effect against azole-resistant fungi. In YPD solid medium, this synergistic effect is significantly related to the dose-response relationship of azole drugs.

[0087] Example 4

[0088] Determination of inhibition zone of NIM combined with FLC against FLC-resistant Candida albicans

[0089] Materials and methods

[0090] 1. Medicinal materials: Same as in Examples 1 and 2.

[0091] 2. Strains: Candida albicans Y0109 and 9821 were provided by the Department of Dermatology, Shanghai Changzheng Hospital; Candida albicans 7654 was provided by the Department of Pharmaceutical Chemistry, Department of Pharmacy, Naval Medical University. The methods for cryopreservation and activation of the strains were the same as in Example 1.

[0092] 3. Culture medium

[0093] The preparation methods for YPD liquid and solid culture media are the same as in Example 1. The final concentrations of NIM in the solid culture media are 2, 4, and 8 μg / mL, respectively.

[0094] 4. Instruments: Same as in Example 3.

[0095] 5. Paper diffusion experiment method

[0096] The experimental method was the same as in Example 3. The experimental results are shown below. Figure 2 As shown, Figure 2 This is a schematic diagram showing the results of the determination of the inhibition zone of resistant fungi when NIM and FLC are used together in Example 4.

[0097] The results showed that: (1) For FLC-resistant Candida albicans Y0109, in the Control group (without NIM), a clear inhibition zone was formed around the FLC-loaded filter paper on the agar plate after 24 h of culture. However, after 48 h of culture, a large number of colonies grew in the inhibition zone, and the boundary of the inhibition zone became blurred. In contrast, after 48 h of culture, no bacterial strains grew in the inhibition zone around the filter paper on plates containing different concentrations (2, 4, 8 μg / mL) of NIM. The boundary of the inhibition zone was clear, and the inhibition zone showed a trend of increasing with the increase of FLC loading. Figure 2 As shown in A, the sample addition diagram is shown in C in 2); (2) For FLC-resistant Candida albicans 7654 and 9821, after 48 h of culture, compared with the Control group, the two FLC-resistant bacteria formed obvious inhibition zones around the FLC drug-loaded filter paper on the plate containing 4 μg / mL NIM, and no fungal growth was observed inside the inhibition zones ( Figure 2 As shown in Figure B, and the sample addition diagram is shown in Figure C. The above results indicate that the combined use of NIM and FLC has a significant effect against FLC-resistant Candida albicans.

[0098] Example 5

[0099] Determination of the time-growth curve of NIM combined with FLC against drug-resistant fungi

[0100] Materials and methods

[0101] 1. Medicinal materials: Same as in Examples 1 and 2.

[0102] 2. Strains: Candida albicans 904 was provided by the Department of Dermatology, Shanghai Changzheng Hospital. The cryopreservation and activation methods for the strain were the same as in Example 1.

[0103] 3. Culture medium: Same as in Example 1.

[0104] 4. Instruments: Same as in Example 1.

[0105] 5. Determination of time-growth curve

[0106] Candida albicans was inoculated into YPD liquid medium and cultured at 30 °C for 16 h. The activated bacterial suspension was transferred to a 1.5 mL centrifuge tube, centrifuged at 3000 g for 1 min, the medium was discarded, and the cells were washed twice with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1 mL PBS. After dilution, 10 μL was transferred to a hemocytometer for counting. The bacterial concentration was adjusted to 1 × 10⁻⁶ cells / mL using RPMI 1640 liquid medium. 6 The bacterial culture was divided into several portions and transferred to 50 mL centrifuge tubes, 10 mL per tube. Different concentrations (2, 4, 8, 16 μg / mL) of the test drug NIM were added to each tube. Then, different concentrations (0, 2, 4, 8 μg / mL) of FLC were added to the tubes containing 2 μg / mL NIM, and the same procedure was repeated for the tubes containing 4, 8, and 16 μg / mL NIM. Finally, the remaining four tubes were used: three tubes containing only different concentrations (2, 4, 8 μg / mL) of FLC, and one tube containing an equal volume of DMSO solvent as the drug. All centrifuge tubes containing bacterial culture were incubated at 30 ℃ with shaking at 200 rpm. At 0, 4, 8, 12, and 24 h, 100 μL of each bacterial culture was aspirated into a 96-well plate (vortexing the bacterial culture in each tube before sampling, and taking three replicates for each sample) to measure the OD of each well. 630 And graphs were plotted using GraphPad. Experimental results are shown below. Figure 3 As shown, Figure 3 This is a schematic diagram showing the time-growth curve results of the combined use of NIM and FLC to combat drug-resistant fungi in Example 5.

[0107] The results showed that, compared with the control group, NIM alone (2, 4, 8, or 16 μg / mL) had no significant effect on the growth of the strain, while FLC alone (2, 4, or 8 μg / mL) had a certain inhibitory effect on the growth of Candida albicans, but the inhibition rate was less than 50%. When the FLC concentration was fixed at 2, 4, or 8 μg / mL, the inhibitory effect of the two drugs combined on the drug-resistant bacteria gradually increased with the increase of NIM concentration, especially when the NIM concentration reached 16 μg / mL, the combined use with 2, 4, and 8 μg / mL FLC had a significant inhibitory effect on the drug-resistant bacteria (inhibition rate > 80%). These results indicate that in liquid culture medium, NIM can enhance the antifungal activity of FLC, and this enhancement effect is significantly concentration-dependent.

[0108] Example 6

[0109] Determination of the in vivo antifungal effect of NIM combined with FLC

[0110] Materials and methods

[0111] 1. Medicinal materials: Same as in Examples 1 and 2.

[0112] NIM and FLC were prepared into a 51.2 mg / mL solution using DMSO, and the test drugs were stored at -20°C. Before the experiment, the drug stock solution was removed and allowed to thaw at room temperature, thoroughly mixed, diluted, and then used for in vivo experiments.

[0113] The average weight of the wax moth larvae is 0.5 g / larva, and the body fluid is calculated at 100 μL / larva. The drug solution administered to the wax moth larvae is diluted with physiological saline (DMSO content not exceeding 10%) to the appropriate concentration.

[0114] 2. Strains: FLC-resistant Candida albicans 904 was provided by the Department of Dermatology, Shanghai Changzheng Hospital. The cryopreservation and activation methods for the strain were the same as in Example 1.

[0115] 3. Culture medium: Same as in Example 1.

[0116] 4. Animals: The wax moth larvae used in the experiment were purchased from Crawling Vision Company, weighing approximately 0.5 g.

[0117] 5. Instruments: Biosafety cabinet (BSC-1004II A2, Suzhou Antai Air Technology Co., Ltd.); biological microscope (LW100T, Beijing Cewei Optoelectronic Technology Co., Ltd.); constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd. MJ-150-I); shaking incubator (Jiangsu Taicang Experimental Equipment Factory HZ-2111K-B); small centrifuge (Hitachi CT15RE).

[0118] 6. In vivo experimental methods

[0119] Candida albicans infection model in wax moth: Candida albicans 904 was inoculated into YPD liquid medium and cultured at 30℃ for 16 h. The activated bacterial suspension was transferred to a 1.5 mL centrifuge tube, centrifuged at 3000 g for 1 min, the medium was discarded, and the cells were washed twice with PBS. After centrifugation, the supernatant was discarded, and the suspension was resuspended in 1 mL PBS. After dilution, 10 μL was transferred to a hemocytometer for counting. The bacterial concentration was adjusted to 1×10⁻⁶ cells / mL with sterile PBS. 8 cells / mL. Sixteen healthy wax moth larvae of similar size were placed in the same petri dish, and the average weight of the larvae was calculated. 5 μL of bacterial suspension was injected into the last leg on the left side of the larvae. In the model group, 5 μL of the solvent used to dissolve the drug was randomly injected into the last leg on the right side. Each drug-treated group received 5 μL of different concentrations of NIM, FLC, or a combination of both drugs injected into the last leg on the right side. Syringes were cleaned with ethanol and water before and after each use. After injection, the wax moth larvae were incubated at 37℃. Mortality was observed and recorded daily for 10 consecutive days. Survival curves were plotted, and Log-rank statistical analysis was performed using GraphPad Prism software. The experimental results are shown below. Figure 4 As shown, Figure 4 This is a schematic diagram showing the results of Example 6, in which the combined use of NIM and FLC effectively improved the survival rate of wax moth larvae infected with drug-resistant Candida albicans.

[0120] Experimental Results: Within 3 days of Candida albicans infection, all wax moth larvae in the model group died. At the end of the 10-day observation period, the survival rate of the NIM (0.4 mg / kg) monotherapy group was only 12.5%, and the survival rates of the low (0.2 mg / kg), medium (0.4 mg / kg), and high (0.8 mg / kg) FLC monotherapy groups were only 6.25%, 6.25%, and 25%, respectively. However, the combined NIM and FLC treatment at low (NIM 0.4 mg / kg + FLC 0.2 mg / kg), medium (NIM 0.4 mg / kg + FLC 0.4 mg / kg), and high (NIM 0.4 mg / kg + FLC 0.8 mg / kg) doses increased the survival rate of wax moth larvae to 18.75%, 50%, and 81.25%, respectively. The survival rates of the medium and high dose groups in the combined treatment were significantly higher than those in the FLC monotherapy group. The significance was determined using the Log-rank test, and the results showed that compared with the treatment group treated with FLC alone at medium and high doses, the combined treatment group treated with both drugs at medium and high doses significantly improved the survival rate of infected wax moth larvae (*). < 0.05, ** < 0.01). The above results indicate that NIM exhibits the effect of an antifungal synergist of FLC in vivo.

[0121] Example 7

[0122] NIM and FLC combined mammalian cytotoxicity assay

[0123] Materials and methods

[0124] 1. Medicinal materials: Same as in Examples 1 and 2. CCK-8: Purchased from Shanghai Taoshu Biotechnology Co., Ltd.

[0125] 2. Cell line: Mouse mononuclear macrophages (RAW264.7) were purchased from Pronosei. The RAW cells used in the experiment were cultured in complete medium (DMEM + 10% fetal bovine serum) at 37°C.

[0126] 3. Culture media: DMEM high-glucose medium was purchased from Shanghai Titan Technology Co., Ltd. Fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd.

[0127] 4. Instruments: Biosafety cabinet (BSC-1004II A2, Suzhou Antai Air Technology Co., Ltd.); 37℃ cell culture incubator (MCO-170AICDL-PC, Puhexi Health Medical Equipment Co., Ltd.); Multifunctional microplate reader (TECAN InfiniteM200).

[0128] 5. Cytotoxicity assay methods

[0129] Mouse mononuclear macrophages (RAW264.7) cultured to approximately 80% confluence were removed from the 37°C incubator. The culture medium was discarded, and the cells were washed twice with 2 mL of PBS. The cells were then mixed with 3 mL of PBS and transferred to a 15 mL centrifuge tube. The cells were centrifuged at 1000 rpm for 3 min, and the supernatant was discarded. The cells were resuspended in 3 mL of DMEM medium containing 10% fetal bovine serum and 2% penicillin-dextrose antibiotics to obtain a cell suspension. 100 μL of this cell suspension was diluted 10-fold in 900 μL of complete culture medium. 10 μL of the cell dilution was transferred to a cell counting chamber for cell counting. The cell density was then adjusted to 1 × 10⁻⁶ cells / mL. 5Cells / mL. Divide a 96-well plate into experimental, control, and cell-free zones. Add 100 μL of cell culture to each well in the experimental and control zones, and 100 μL of complete culture medium to each well in the cell-free zone. Ensure even cell distribution within the wells. Incubate the cells overnight at 37 ℃. The next day, prepare a drug-addition plate using a sterile 96-well plate. Based on the survival rate experiment of wax moth larvae, calculate the optimal drug dosage (NIM 0.4 mg / kg + FLC 0.8 mg / kg). The average weight of a wax moth larva is 0.5 g / larva, and its body fluid is approximately 100 μL / larva. Therefore, NIM 0.4 mg / kg + FLC 0.8 mg / kg is equivalent to a drug concentration of NIM 2 μg / mL + FLC 4 μg / mL. To verify safety, a 16-fold amplification was designed based on this concentration. This involved preparing complete medium containing 32 μg / mL NIM + 64 μg / mL FLC, and then serially diluting the complete medium to 2 μg / mL NIM + 4 μg / mL FLC. The 96-well cell culture plates were then removed from the incubator, and the original medium from the experimental and control areas was discarded. 100 μL of medium containing different drug concentrations (triple replicates for each concentration) was added to each well, and the plates were incubated at 37°C for 24 h. The 96-well plates were then removed, and the original medium from the experimental, control, and cell-free areas was discarded. 110 μL of complete medium containing CCK-8 (CCK-8:complete medium = 1:10) was added to each well, and the plates were incubated at 37°C for 2 h. The OD values ​​of each well were measured. 450 When processing the results, the OD value of the cell-free area was subtracted from the OD value of the experimental and control areas, and the Cell Viability of the control area was set to 1.0 to calculate the Cell Viability of each group. The experiment was repeated three times, and graphs were plotted and statistically analyzed using GraphPad. The experimental results are shown below. Figure 5 , Figure 5 This is a schematic diagram of the cytotoxicity results of the combined use of NIM and FLC in Example 7, where the horizontal axis represents the concentration of NIM and FLC (µg / mL) and the vertical axis represents cell viability (%).

[0130] The results showed that when 32 μg / mL NIM was used in combination with 64 μg / mL LFLC, the cell viability was 74%, which was not statistically different from the growth control group. >0.05). This concentration is 16 times the optimal dose for the combined use of the two drugs in the wax moth larvae survival experiment, indicating that the antifungal effect of NIM and FLC is specific and has low toxicity to mammalian cells.

[0131] Example 8

[0132] NIM and FLC combined for toxicity testing of wax moth larvae

[0133] Materials and methods

[0134] 1. Medicinal materials: The source and preparation method of the medicinal materials are the same as in Example 6.

[0135] 2. Animals: Same as in Example 6.

[0136] 3. Instruments: Same as in Example 6.

[0137] 4. In vivo toxicity test methods

[0138] Sixteen healthy wax moth larvae of similar size were placed in the same petri dish, and the average weight of the larvae was calculated. 5 μL of PBS solution was injected into the last leg on the left side of each larva. The Sham group immediately received 5 μL of the drug dissolving solvent in the last leg on the right side. The drug-treated groups received 5 μL of different concentrations of NIM and FLC combinations (NIM 0.4 mg / kg + FLC 0.8 mg / kg, NIM 1.6 mg / kg + FLC 3.2 mg / kg, NIM 6.4 mg / kg + FLC 12.8 mg / kg) in the last leg on the right side. Syringes were cleaned with ethanol and water before and after each use. After injection, the wax moth larvae were incubated at 37°C. Mortality was observed and recorded daily for 10 consecutive days. Survival curves were plotted, and Log-rank statistical analysis was performed using GraphPad Prism software. The results are as follows: Figure 6 As shown, Figure 6 This is a schematic diagram of the toxicity results of NIM and FLC used in combination for wax moth larvae in Example 8.

[0139] The results showed that the combined use of NIM (6.4 mg / kg) and FLC (12.8 mg / kg) resulted in no mortality in wax borer larvae, demonstrating high safety. This dose was 16 times the optimal dose for treating fungal infections in wax borer larvae with the two drugs combined. This indicates that the combined use of NIM and FLC is highly safe for wax borer larvae.

[0140] Example 9

[0141] Determination of inhibition zones of nonsteroidal anti-inflammatory drugs (NSAIDs) in combination with FLC against drug-resistant Candida albicans.

[0142] Materials and methods

[0143] 1. Reagents: NIM, Rofecoxib, Meloxicam, Etoricoxib, Aspirin, Indomethacin, Ibuprofen, and Flurbiprofen were all purchased from Macklin Pharmaceuticals; FLC was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; and Dimethyl sulfoxide (DMSO) was purchased from Sinopharm Chemical Reagent Co., Ltd. NIM, Rofecoxib, Meloxicam, Etoricoxib, Aspirin, Indomethacin, Ibuprofen, and Flurbiprofen were prepared into 10 mg / mL solutions using DMSO. The test drugs were stored at -20°C. Before the experiment, the drug stock solutions were removed and thawed at room temperature, thoroughly mixed, and then used for subsequent experiments.

[0144] 2. Strains: FLC-resistant Candida albicans 904 was provided by the Department of Dermatology, Shanghai Changzheng Hospital. The cryopreservation and activation methods for the strain were the same as in Example 1.

[0145] 3. Culture medium: Same as in Example 3. The concentration of NSAIDs in the solid culture medium was 16 μg / mL.

[0146] 4. Instruments: Same as in Example 3.

[0147] 5. Paper diffusion experiment procedure

[0148] The experimental method was the same as in Example 3. The experimental results are shown below. Figure 7 , Figure 7 This is a schematic diagram showing the results of the inhibition zone determination of drug-resistant fungi when nonsteroidal anti-inflammatory drugs are used in combination with FLC in Example 9.

[0149] The results showed that, within the drug concentration range used in the experiment, compared with the control agar plates without NSAIDs, some agar plates containing NSAIDs (16 μg / mL) showed inhibition zones of varying sizes and clarity around the drug-loaded filter paper containing FLC. This invention investigated the effects of various nonsteroidal anti-inflammatory drugs (NSAIDs) combined with FLC on FLC-resistant Candida albicans. The tested NSAIDs included non-selective COX inhibitors such as aspirin, indomethacin, ibuprofen, and flurbiprofen, and selective COX2 inhibitors such as NIM, rofecoxib, meloxicam, and etoricoxib. NIM showed a strong synergistic effect on FLC, while rofecoxib, meloxicam, etoricoxib, and aspirin had no synergistic effect on FLC. Indomethacin, ibuprofen, and flurbiprofen showed only a weak synergistic effect when combined with FLC. The IC50 values ​​of the potent COX inhibitor indomethacin for COX1 and COX2 were also discussed. 50 The values ​​were 18 nM and 26 nM, respectively, indicating that the IC50 of the selective and potent COX2 inhibitor rofecoxib against COX2 was... 50The value is 18~26 nM, while NIM corresponds to the IC of COX2. 50 The value was 40 nM. Surprisingly, the synergistic antifungal activity of NIM and FLC against drug-resistant fungi was superior to that of the potent COX2 inhibitors indomethacin and rofecoxib. These results suggest that the synergistic antifungal mechanism of NIM and FLC is independent of their COX2 inhibitory effects.

[0150] Example 10

[0151] Effects of the arachidonic acid pathway on the antifungal activity of NIM combined with FLC

[0152] Materials and methods

[0153] 1. Reagents: NIM, prostaglandin E2 (PGE2), and arachidonic acid (AA) were purchased from Macklin Biotechnology Co., Ltd.; FLC was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; dimethyl sulfoxide (DMSO) was purchased from Sinopharm Chemical Reagent Co., Ltd. NIM and FLC were prepared into 10 mg / mL solutions using DMSO, and PGE2 was prepared into 1 mg / mL solutions using DMSO. The test drugs were stored at -20°C. Before the experiment, the drug stock solutions were removed and thawed at room temperature, thoroughly mixed, and then used for subsequent experiments.

[0154] 2. Strains: FLC-resistant Candida albicans 904 was provided by the Department of Dermatology, Shanghai Changzheng Hospital. The cryopreservation and activation methods for the strain were the same as in Example 1.

[0155] 3. Culture medium: Same as in Example 3. The solid culture medium contains different concentrations of NIM, PGE2, or AA.

[0156] 4. Instruments: Same as in Example 3.

[0157] 5. Paper diffusion experiment procedure

[0158] The experimental method was the same as in Example 3. 100 μL of diluted bacterial suspension was poured onto agar plates containing different concentrations (8, 16, 32, 64 μg / mL) of NIM, different concentrations (10, 50, 250 ng / mL) of PGE2, different concentrations (75, 150, 300 μg / mL) of AA, or NIM + PGE2, NIM + AA. The bacterial suspension was spread evenly with sterile glass beads. Sterile filter paper discs (6 mm in diameter) were placed on solid culture media, and an equal volume (5 μL) of different concentrations (0.156–5 mg / mL) of FLC was added to the center of the disc. The agar plates were inverted and incubated at 30°C. The formation of inhibition zones was recorded by photographing at 24 h and 48 h.

[0159] The experimental results are shown in Figure 8 , 9 As shown, Figure 8 This is a schematic diagram illustrating the effect of PGE2 on the combined use of NIM and FLC against drug-resistant fungi in Example 10. The results show that on agar plates containing only PGE2, no inhibition zone was formed around the drug-loaded filter paper containing FLC. On agar plates containing only 16 μg / mL NIM, a clearly defined inhibition zone formed around the drug-loaded filter paper containing FLC, and its size increased with the increase of FLC content on the filter paper. Compared to agar plates containing only NIM, the size of the inhibition zone formed around the drug-loaded filter paper did not change on agar plates containing NIM culture medium with different concentrations (10, 50, 250 ng / mL) of PGE2.

[0160] Figure 9 This is a schematic diagram showing the effect of AA on the combined use of NIM and FLC against drug-resistant fungi in Example 10. The results show that, compared with agar plates containing only NIM, the inhibition zone formed around the drug-loaded filter paper of FLC on agar plates containing NIM medium with different concentrations (75, 150, 300 μg / mL) of AA decreased with increasing AA concentration. That is, the addition of AA weakened the synergistic antifungal effect of the two drugs.

[0161] The results of Examples 9 and 10 indicate that the synergistic antifungal effect of NIM combined with FLC is not caused by the COX2 inhibition of NIM (COX2 inhibition reduces the product PGE2 in the arachidonic acid pathway, leading to the accumulation of substrate AA).

[0162] Example 11

[0163] Comparison query of human cyclooxygenase 2 (COX2) homologous genes or proteins in Candida albicans

[0164] 1. Sequence alignment tools

[0165] NCBI official website: https: / / www.ncbi.nlm.nih.gov /

[0166] 2. Sequence alignment methods

[0167] Open the NCBI website and search for the nucleic acid or amino acid sequence of human cyclooxygenase 2 (COX2). On the NCBI homepage, click on the BLAST tool to enter the main page. Choose the appropriate BLAST tool based on the type of sequence you want to align (nucleotide BLAST for nucleic acid sequences, protein BLAST for protein sequences). Enter the nucleic acid or amino acid sequence in the corresponding field on the BLAST interface, select the database to align to (usually standarddatabases by default), and select the species to align to (here, select...). In Program Selection, the default setting is Highly similar sequences. Click blast, and wait about a minute on the redirected page until the information page pops up.

[0168] The comparison results are shown below. Figure 10 As shown, Figure 10 This is a schematic diagram showing the alignment results of human COX2 homologous genes or proteins in *Candida albicans* in Example 11. In this diagram, A shows the alignment results of human COX2 homologous genes in *Candida albicans*, and B shows the alignment results of human COX2 homologous proteins in *Candida albicans*. The results show that no genes or proteins with significant homology to human cyclooxygenase 2 (COX2) were found in *Candida albicans*. These results suggest that the synergistic antifungal mechanism of NIM and FLC does not depend on COX2 inhibition.

[0169] Example 12

[0170] The effect of thin-layer chromatography on the combined use of NIM and FLC on ergosterol content

[0171] Materials and methods

[0172] 1. Medicinal materials: Same as in Example 1. Ergosterol, lanosterol, and squalene were purchased from Macklin Company; petroleum ether (boiling range 30-60℃), cyclohexane, and ethyl acetate were purchased from Shanghai Titan Technology Co., Ltd.

[0173] 2. Strains: Candida albicans 904 was provided by the Department of Dermatology, Shanghai Changzheng Hospital. The cryopreservation and activation methods for the strain were the same as in Example 1.

[0174] 3. Culture medium: Same as in Example 1.

[0175] 4. Instruments: Biosafety cabinet (BSC-1004II A2, Suzhou Antai Air Technology Co., Ltd.); biological microscope (LW100T, Beijing Cewei Optoelectronic Technology Co., Ltd.); constant temperature incubator (MJ-150-I, Shanghai Yiheng Scientific Instrument Co., Ltd.); shaking incubator (HZ-2111K-B, Jiangsu Taicang Experimental Equipment Factory); vortex apparatus (Thermo Vortex Maximix II); mini centrifuge (Hitachi CT15RE).

[0176] 5. Experimental Procedure

[0177] Take five 250 mL Erlenmeyer flasks and add 150 mL of YPD medium. Transfer 1.5 mL of activated drug-resistant Candida albicans 904 to each of the five Erlenmeyer flasks. Add FLC (2, 4 μg / mL), NIM (2, 4 μg / mL), or FLC (2 or 4 μg / mL) + NIM (2 or 4 μg / mL) to each flask, respectively. For the blank control, add an equal volume of DMSO solvent as the drug-treated group. Incubate at 30 °C and 200 rpm for 16 h with shaking. After the bacterial culture is complete, centrifuge at 3000 g for 5 min and wash three times with PBS until the supernatant is colorless. Discard the supernatant, weigh 1.0 g of each bacterial strain, place it in an 80 mL unstoppable glass tube, add 5 mL of PBS buffer and 12 mL of freshly prepared saponifying agent (90% ethanol solution of 15% NaOH), mix well, and saponify in an 80 °C water bath for 60 min. Cool to room temperature. Add 10 mL of petroleum ether (boiling range 30-60 ℃), vortex for 1 min, extract three times, combine the extracts, add 10 mL of water, vortex and wash, remove the aqueous layer (let the pipette tip down for a moment, the turbid liquid is the aqueous layer and should be discarded). Evaporate the ether layer in a 60 ℃ water bath (approximately 1.5 h) to obtain unsaponified lipids, add 0.9 mL of cyclohexane, mix well, seal with plastic wrap, and store at 4 ℃ for later use. Thin-layer chromatography conditions: saturated vapor with cyclohexane (cyclohexane:ethyl acetate = 4:1) for 30 min, develop to 8 cm; 10% sulfuric acid ethanol solution spray, develop at 110 ℃ for 60 min. Spot 10 μL of the reference standard and various sample solutions onto a high-performance silica gel plate for development and color development.

[0178] Figure 11This is a schematic diagram of the thin-layer chromatography results of the effect of NIM combined with FLC on ergosterol content in Example 12. The results showed that: (1) Compared with the Control group of drug-resistant bacteria 904, the NIM-only group had no effect on sterol content, while the FLC-only group showed a decrease in ergosterol content and an increase in lanosterol and squalene content, which were dose-dependent with FLC. (2) Compared with FLC alone, the combination of NIM and FLC had no significant difference in the sterol components such as ergosterol and lanosterol in the ergosterol synthesis pathway. The above results suggest that the synergistic antifungal effect of NIM and FLC is not achieved by affecting the ergosterol synthesis pathway.

[0179] Example 13

[0180] Effect of combined gas chromatography-mass spectrometry (GC-MS) on the effect of NIM and FLC on the sterol components of Candida albicans

[0181] Materials and methods

[0182] 1. Herbal materials: Same as in Example 12. Cholesterol was purchased from Sangon Biotech (Shanghai) Co., Ltd., and n-hexane was purchased from Shanghai Titan Technology Co., Ltd.

[0183] 2. Strains: Candida albicans 904 was provided by the Department of Dermatology, Shanghai Changzheng Hospital. The cryopreservation and activation methods for the strain were the same as in Example 1.

[0184] 3. Culture medium: Same as in Example 1.

[0185] 4. Instruments: Biosafety cabinet (BSC-1004II A2, Suzhou Antai Air Technology Co., Ltd.); biological microscope (LW100T, Beijing Cewei Optoelectronic Technology Co., Ltd.); constant temperature incubator (MJ-150-I, Shanghai Yiheng Scientific Instrument Co., Ltd.); shaking incubator (HZ-2111K-B, Taicang Experimental Equipment Factory, Jiangsu Province); vortex apparatus (Thermo Vortex Maximix II); multi-functional microplate reader (BioTek Synergy H1); DSQ II / Trace GC Ultra gas chromatography-mass spectrometry system (Thermo Scientific).

[0186] 5. Experimental Procedure

[0187] Experimental condition setting (time-growth curve): The experimental method was the same as before. The bacterial concentration was adjusted to 1×10⁻⁶ using YPD liquid medium. 6The bacterial culture was divided into several portions and transferred to 50 mL centrifuge tubes, 10 mL per tube. Six tubes of bacterial culture were then added with different concentrations (1, 4, 16 μg / mL) of the test drug NIM, three tubes per group, for a total of two groups. In one group, 16 μg / mL of FLC was added to the three tubes. Two other tubes of bacterial culture were taken; one tube was added with FLC (16 μg / mL), and the other with an equal volume of DMSO as the drug solution. All bacterial cultures were placed in a 30 ℃ constant temperature shaking incubator and cultured at 200 rpm. At 0, 8, 16, and 24 h, 100 μL of each bacterial culture was aspirated into a 96-well plate (vortexed to mix the bacterial culture before sampling, three replicates per sample). The OD630 of each well was measured and plotted using GraphPad. The experimental results are shown below. Figure 12 As shown.

[0188] GC-MS detection of sterol components: Take several 250 mL Erlenmeyer flasks and add 150 mL of YPD medium. Transfer 1.5 mL of activated, drug-resistant Candida albicans 904 to each Erlenmeyer flask. Add FLC, NIM, and FLC+NIM respectively, and add an equal volume of DMSO to the blank control to make the final drug concentrations 16 μg / mL for FLC, 16 μg / mL for NIM, and 16 μg / mL for FLC + 16 μg / mL for NIM. Incubate at 30 °C and 200 rpm for 16 h with shaking. After the bacterial culture is complete, centrifuge at 3000 g for 5 min, and wash three times with PBS until the supernatant is colorless. Discard the supernatant, accurately weigh approximately 1.0 g of each bacterial strain, place it in an 80 mL unstoppable glass tube, add 10 mL of saponifying agent (90% ethanol solution containing 15% NaOH), mix well, saponify in an 80 °C water bath for 60 min, and cool to room temperature. Then, 2 mL of ultrapure water was added to each sample, and the samples were extracted twice with 6 mL of n-hexane. The organic phases were combined and washed with ultrapure water. The organic phase was then separated and dried under vacuum or allowed to evaporate naturally. 1 mL of cyclohexane was added to the evaporated residue to dissolve it. For content determination, 390 μL of each sample tube was pipetted, and 10 μL of 4 mg / mL cholesterol solution (prepared in cyclohexane) was added as an internal standard. The samples were centrifuged (11000 rpm, 3 min), and the supernatant was injected for GC-MS analysis. The chromatographic column was DB-5ms (30 m × 0.25 mm × 0.25 μm). The mass spectrometry results were compared with the NIST database to obtain possible sterol structures. Experimental results are shown below. Figure 13 Table 3 Figure 14 As shown.

[0189] Figure 12This is a schematic diagram showing the results of the experiment in Example 13 exploring the drug concentration and treatment time for GC-MS detection of sterol components. The results showed that after 16 h of drug treatment, there were significant differences in the growth of Candida albicans treated with FLC 16 μg / mL, NIM 16 μg / mL, and FLC 16 μg / mL + NIM 16 μg / mL. Therefore, this condition was selected for subsequent experiments.

[0190] Figure 13 Table 3 shows the gas chromatograms of various sterol components of Candida albicans after drug treatment detected by GC-MS in Example 13; Table 3 shows the results of GC-MS determination of the effect of the combined use of NIM and FLC on the proportion of various sterol components of Candida albicans in the total sterols in Example 13. Figure 14 This is a schematic diagram of the effect of GC-MS detection of the combined use of NIM and FLC on the proportion of each sterol component in the total sterols of Candida albicans in Example 13. The results showed that: (1) In the Control group, ergosterol accounted for 90.193%, and the proportions of Obtusifoliol and Eburicol in the sterol synthesis salvage pathway were both less than 0.5%; (2) In the FLC 16 μg / mL group, Erg11p was inhibited, and sterol synthesis was mainly carried out through the salvage pathway, with Eburicol accounting for 80.576% in the salvage pathway; (3) In the NIM 16 μg / mL group, ergosterol accounted for 92.832%, which was not significantly different from the Control group (90.193%); (4) In the combined use of the two drugs, sterol synthesis was mainly carried out through the salvage pathway, with Eburicol accounting for 79.373% in the salvage pathway, and the composition of each sterol was not significantly different from the FLC 16 μg / mL single use group.

[0191] Table 3

[0192]

[0193] The results of Examples 12 and 13 consistently indicate that NIM's role as an agonist for azole antifungal drugs is not through affecting the ergosterol synthesis pathway.

[0194] Example 14

[0195] Determination of the effect of NIM on the drug efflux pump of Candida albicans

[0196] Materials and methods

[0197] 1. Reagents: NIM and kaempferol were purchased from Macklin Chemical Co., Ltd., Rhodamine 6G (R6G) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and dimethyl sulfoxide (DMSO) was purchased from Sinopharm Chemical Reagent Co., Ltd. NIM and kaempferol were prepared into 10 mg / mL solutions using DMSO, and Rhodamine 6G was prepared into a 10 mM solution using DMSO. The test drugs were stored at -20°C. Before the experiment, the drug stock solutions were removed and allowed to thaw at room temperature, then thoroughly mixed before subsequent experiments.

[0198] 2. Strains: Candida albicans SC5314 was purchased from Georgetown University; FLC-resistant Candida albicans 901 and 953 were provided by the Department of Pharmaceutical Chemistry, Department of Pharmacy, Naval Medical University; and FLC-resistant Candida albicans 904 and 632 were provided by the Department of Dermatology, Shanghai Changzheng Hospital. The methods for cryopreservation and activation of the strains were the same as in Example 1.

[0199] 3. Culture medium: Same as in Example 1.

[0200] 4. Instruments: Same as in Example 1.

[0201] 5. Experimental procedures for determining the efflux capacity of Rhodamine 6G

[0202] The test strain was inoculated into YPD liquid medium and cultured at 30°C for 16 h. The activated bacterial suspension was transferred to a 1.5 mL centrifuge tube, centrifuged at 3000 g for 1 min, the medium was discarded, and the cells were washed twice with PBS buffer. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1 mL PBS. After dilution, 10 μL was transferred to a hemocytometer for counting. The bacterial suspension concentration was adjusted to 5 × 10⁻⁶ cells / mL with PBS buffer. 7 Cells / mL. Incubate at 30℃ and 200 r / min for 2 h with shaking to fully deplete cellular energy. Resuspend the treated Candida albicans and adjust the bacterial concentration to 5 × 10⁻⁶ cells / mL. 7 The cells were collected at a concentration of 10 cells / mL and divided into groups of 10 mL each. Each group was then incubated with 10 μL of R6G stock solution (final concentration 10 μmol / L) at 200 rpm for 1 h at 30°C to reach R6G uptake equilibrium. The cells were then placed on ice for 10 min to stop R6G uptake. After centrifugation at 5000 rpm for 5 min, the cells were collected and washed three times with glucose-free PBS buffer to remove unabsorbed R6G. The cells were resuspended in PBS containing 5% glucose (final concentration 20 mmol / L) to achieve a bacterial concentration of 5 × 10⁻⁶ cells / mL. 7The experimental groups investigating the effect of drugs on efflux pumps were set up as follows: test drug group (NIM 8 μg / mL), positive control drug group (kaempferol 8 μg / mL), and solvent control group (DMSO added in an equal volume to the drug solution). Each group of bacterial cultures was cultured at 30℃ with shaking at 200 r / min. Samples of 1 mL were taken at 0 min, 30 min, 60 min, and 120 min, centrifuged at 10000 rpm for 1 min, and the supernatant was carefully transferred to black 96-well plates, with 3 replicates per sample and 100 μL per well. The fluorescence value of the supernatant was detected using a microplate reader (excitation wavelength 488 nm, emission wavelength 530 nm).

[0203] See results Figure 15 As shown, Figure 15 This is a schematic diagram illustrating the effect of NIM on the drug efflux pump of Candida albicans in Example 14. In Example A, the drug efflux capacity of clinically FLC-resistant Candida albicans 904, 901, 632, and 953 was examined. In Example B, the Candida albicans 632, with the strongest efflux capacity, was selected as the research subject to investigate the effect of drug treatment on the efflux capacity of Candida albicans. The results showed that compared with the Control group, the efflux pump inhibitor kaempferol (8 μg / mL) significantly inhibited the rhodamine 6G efflux capacity of Candida albicans, while NIM (8 μg / mL) had no significant effect on the rhodamine 6G efflux capacity of Candida albicans. These results indicate that the effect of NIM as an antifungal drug synergist is not produced by inhibiting the drug efflux pump of Candida albicans.

[0204] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The use of nimesulide or its pharmaceutical salts in combination with azole antifungal drugs or allylamine antifungal drugs in the preparation of antifungal drugs, characterized in that, The azole antifungal drugs are selected from fluconazole, itraconazole, and voriconazole; The allylamine antifungal drug is selected from terbinafine.

2. The use of nimesulide or its pharmaceutical salts combined with azole antifungal drugs or allylamine antifungal drugs according to claim 1 in the preparation of antifungal drugs, characterized in that, The medicinal salts referred to are acid addition salts formed by nimesulide with the following acids: hydrochloric acid, sulfuric acid, nitric acid, citric acid, and maleic acid.

3. The use of nimesulide or its pharmaceutical salts combined with azole antifungal drugs or allylamine antifungal drugs according to claim 1 in the preparation of antifungal drugs, characterized in that, The fungi are selected from Candida albicans and Candida tropicalis.

4. The use of nimesulide or its pharmaceutical salts combined with azole antifungal drugs or allylamine antifungal drugs according to claim 3 in the preparation of antifungal drugs, characterized in that, For fluconazole-resistant Candida albicans, the combined treatment regimen is: nimesulide concentration of 1~32 μg / mL + fluconazole concentration of 0.5~16 μg / mL.

5. The use of nimesulide or its pharmaceutical salts combined with azole antifungal drugs or allylamine antifungal drugs according to claim 3 in the preparation of antifungal drugs, characterized in that, For fluconazole-resistant Candida albicans, the combined treatment regimen is: nimesulide at a concentration of 2-4 μg / mL + itraconazole at a concentration of 0.25-1 μg / mL.

6. The use of nimesulide or its pharmaceutical salts combined with azole antifungal drugs or allylamine antifungal drugs according to claim 3 in the preparation of antifungal drugs, characterized in that, For fluconazole-resistant Candida albicans, the combined treatment regimen is: nimesulide at a concentration of 2~8 μg / mL + voriconazole at a concentration of 0.25~0.5 μg / mL.

7. The use of nimesulide or its pharmaceutical salts combined with azole antifungal drugs or allylamine antifungal drugs according to claim 3 in the preparation of antifungal drugs, characterized in that, For fluconazole-resistant Candida albicans, the combined treatment regimen is: nimesulide at a concentration of 32~128 μg / mL + terbinafine at a concentration of 4~128 μg / mL.

8. The use of nimesulide or its pharmaceutical salts combined with azole antifungal drugs or allylamine antifungal drugs according to claim 3 in the preparation of antifungal drugs, characterized in that, For fluconazole-resistant Candida tropicalis, the combined treatment regimen is: nimesulide at a concentration of 2~32 μg / mL + fluconazole at a concentration of 16~64 μg / mL.

9. A pharmaceutical preparation, characterized in that, It is made from nimesulide or its pharmaceutical salts combined with azole antifungal drugs or allylamine antifungal drugs.

10. The use of nimesulide or its pharmaceutical salt in the preparation of synergists for azole antifungal drugs or allylamine antifungal drugs.

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