Use of an azole in the preparation of an antifungal

Abstract

The application relates to the application of azole drugs in the preparation of antifungal drugs and belongs to the technical field of antifungal drug research and development. The application of glycyrrhetic acid combined with azole drugs in the preparation of antifungal drugs discloses that glycyrrhetic acid combined with itraconazole, voriconazole and posaconazole shows good synergistic effect or antagonistic effect on aspergillus, exophiala dermatiditis, candida and new cryptococcus, and provides a research and development direction for preparing drugs for resisting aspergillus, exophiala dermatiditis, candida and new cryptococcus.

Description

Technical Field

[0001] This invention relates to the application of azole drugs in the preparation of antifungal drugs, and belongs to the field of antifungal drug research and development technology. Background Technology

[0002] Fungal infections affect more than one billion people annually, ranging from superficial fungal infections to life-threatening invasive fungal diseases, posing a serious threat to global public health. Invasive fungal diseases are of great concern due to their high morbidity and mortality rates, especially in immunocompromised or suppressed individuals, such as those with hematologic malignancies, AIDS, or solid organ transplants. It is estimated that more than 2.5 million people die from invasive fungal diseases each year, more than three times the number from malaria. Approximately 90% of invasive fungal infections are caused by Candida, Cryptococcus, and Aspergillus species, and the limited range of antifungal drugs currently available (polyenes, azoles, echinocandins, and 5-fluorocytosine), coupled with the continuous emergence of drug-resistant fungal strains, makes the treatment of invasive fungal diseases extremely challenging. Among these, Candida is the most common cause of invasive fungal diseases, with Candida albicans being the leading cause. However, infections caused by non-Candida albicans (such as Candida glabrata, Candida parapsilosis, Candida tropicalis, and Candida auris) are increasingly common, accounting for up to 85% of fungal infections in intensive care units and with a mortality rate of approximately 40%-60%. The most common pathogen of invasive aspergillosis, *Aspergillus fumigatus*, has a mortality rate as high as 80% in immunocompromised patients. Other common pathogens include *Aspergillus flavus*, *Aspergillus niger*, and *Aspergillus terreus*. *Cryptococcus neoformans* and *Dark neurotrophic dermatitis dermatitis fungus* can also cause infections with high mortality rates. The former is often found in trees or soil contaminated with bird droppings, while the latter is often found in artificially created hot and humid environments such as saunas and steam rooms. Furthermore, invasive fungal infections impose a considerable economic burden, costing hundreds of millions of dollars annually in the United States alone, and this figure is increasing year by year. Existing antifungal drugs are ineffective against invasive fungal infections, and there is an urgent need to find new drug treatments for these infections. Summary of the Invention

[0003] The purpose of this invention is to provide the application of glycyrrhetinic acid in combination with itraconazole, voriconazole, posaconazole and fluconazole in the preparation of drugs against Aspergillus, Corydalis dermatitis, Candida and Cryptococcus neoformans.

[0004] The technical solution of this invention is:

[0005] The application of an azole drug in the preparation of an antifungal drug is characterized by the use of glycyrrhetinic acid in combination with an azole drug in the preparation of an antifungal drug.

[0006] The azole drug is posaconazole; the antifungal drug is an anti-Aspergillus terrestris drug. The minimum inhibitory concentration (MIC) of glycyrrhetinic acid is >64 µg / mL; the MIC of posaconazole is 0.125-0.25 µg / mL.

[0007] The azole drug is posaconazole; the antifungal drug is an anti-Aspergillus niger, anti-Aspergillus flavus, and anti-Aspergillus fumigatus drug. The minimum inhibitory concentration (MIC) of glycyrrhetinic acid is >64 µg / mL; the MIC of posaconazole is 0.125-1 µg / mL. Glycyrrhetinic acid and posaconazole are mixed together.

[0008] The azole drug mentioned is posaconazole, and the antifungal drug is an anti-dermatitis antifungal drug and an anti-Candida auris drug. The minimum inhibitory concentration (MIC) of glycyrrhetinic acid is >64 µg / mL; the MIC of posaconazole is 0.03125-2 µg / mL. Glycyrrhetinic acid and posaconazole are mixed together.

[0009] The azole drug mentioned is itraconazole, and the antifungal drug mentioned is an anti-Candida glabrata drug, an anti-Candida tropicalis drug, and an anti-Candida auris drug. The minimum inhibitory concentration of glycyrrhetinic acid is >64µg / mL; the minimum inhibitory concentration of itraconazole is 0.0625-8µg / mL.

[0010] The azole drug is itraconazole; the antifungal drug is an anti-cryptococcal drug. The minimum inhibitory concentration (MIC) of glycyrrhetinic acid is >64 µg / mL; the MIC of itraconazole is 0.125-0.25 µg / mL.

[0011] The azole drug is posaconazole; the antifungal drug is an anti-cryptococcal drug. The minimum inhibitory concentration (MIC) of glycyrrhetinic acid is >64 µg / mL; the MIC of itraconazole is 0.125-0.25 µg / mL.

[0012] The glycyrrhetinic acid mentioned above is a pentacyclic triterpenoid derivative of β-amyrin, and is an aglycone produced by the metabolism of glycyrrhizic acid in the human intestine. Glycyrrhetinic acid has a variety of significant biological activities, including anti-inflammatory, antiviral, hepatoprotective, antitumor, autophagy, and apoptosis-inducing effects.

[0013] The advantages of this invention are:

[0014] The application of glycyrrhetinic acid combined with azoles in the preparation of antifungal drugs discloses that glycyrrhetinic acid combined with itraconazole, voriconazole, and posaconazole exhibits good synergistic or antagonistic effects against Aspergillus, Corydalis dermatitis, Candida, and Cryptococcus neoformans, providing a research direction for the preparation of drugs against Aspergillus, Corydalis dermatitis, Candida, and Cryptococcus neoformans. Attached Figure Description

[0015] Figure 1This diagram illustrates the interaction between glycyrrhetinic acid and azole drugs in the anti-Aspergillus reaction of the present invention; A: Synergistic effect of GA in combination with ITC and POS against Aspergillus; B: GA-POS interaction in Aspergillus; C: GA-ITC interaction in Aspergillus; S: Synergistic effect (FICI≤0.5); I: No interaction (0.5<FICI≤4); Synergistic effect = number of strains with synergistic effect / total number of tested strains.

[0016] Figure 2. Illustration of the interaction between glycyrrhetinic acid and azole drugs in combination for treating dermatitis; S: synergistic effect (FICI≤0.5); I: no interaction (0.5<FICI≤4); synergistic rate = number of strains with synergistic effect / total number of tested strains.

[0017] Figure 3. Illustration of the interaction between glycyrrhetinic acid (GA) and azole antifungal drugs on Candida: A: Synergistic and antagonistic rates of GA and azole drugs on Candida; B: GA-POS interaction in Candida; C: GA-ITC interaction in Candida; D: GA-VRC interaction in Candida; E: GA-FLU interaction in Candida; S, synergistic effect (FICI≤0.5); I: no interaction (0.5<FICI≤4); A, antagonistic effect (FICI>4); Synergistic rate = number of strains with synergistic effect / total number of tested strains; Antagonistic rate = number of antagonistic strains / total number of tested strains.

[0018] Figure 4 A diagram illustrating the interaction between glycyrrhetinic acid and azole antifungal drugs on Cryptococcus neoformans; S, synergistic effect (FICI≤0.5); I: no interaction (no interaction, FICI from >0.5 to ≤4); A: antagonistic effect (FICI>4); synergistic rate = number of strains with synergistic effect / total number of tested strains; antagonistic rate = number of antagonistic strains / total number of tested strains. Detailed Implementation

[0019] Given that oleanolic acid, which is structurally highly similar to glycyrrhetinic acid, has been proven to be an effective azole sensitizer, we boldly hypothesize that glycyrrhetinic acid may have a moderating effect on the efficacy of azole drugs. To further explore the potential of the glycyrrhetinic acid-azole combination and provide new insights for antifungal therapy, this experiment, following the M27-A3 protocol issued by the Clinical Laboratory Standards Institute (CLSI), determined the in vitro drug susceptibility of glycyrrhetinic acid with itraconazole, posaconazole, voriconazole, and fluconazole against 107 clinical isolates of Aspergillus, Candida, Epidermophyton, and Cryptococcus neoformans.

[0020] In the following examples or test cases, unless otherwise specified, all raw materials were commercially available. Furthermore, GA represents glycyrrhetinic acid, ITC represents itraconazole, VRC represents voriconazole, POS represents posaconazole, and FLU represents fluconazole.

[0021] 1. In vitro drug susceptibility test of glycyrrhetinic acid combined with triazoles and fluconazole against Aspergillus spp., Epidermophyton floccosum, Candida spp., and Cryptococcus neoformans.

[0022] 1.1. Materials

[0023] 1.1.1 Drugs

[0024] ITC (batch number J2227386, purity ≥ 98%), VRC (batch number H2307623, purity ≥ 98%), POS (batch number H2224157, purity ≥ 98%), FLU (batch number B2216391, purity ≥ 98%), and GA (batch number C2218448, purity = 97%). All drugs were purchased in powder form and dimethyl sulfoxide (DMSO) from Shanghai Aladdin Biochemical Technology Co., Ltd. (China). The powders were dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution (6400 μg / mL).

[0025] 1 .1 .2 strains

[0026] This experiment used a total of 107 clinical fungal isolates, including 56 Aspergillus strains (31 Aspergillus fumigatus, 10 Aspergillus flavus, 10 Aspergillus niger, and 5 Aspergillus terreus), 26 Candida strains (6 Candida albicans, 1 Candida parapsilosis, 3 Candida tropicalis, 6 Candida glabrata, and 10 Candida auris), 16 Erythroderma dermatitidis strains, and 9 Cryptococcus neoformans strains. All isolates were obtained from Jingzhou Central Hospital (Hubei Province, China) and the Hubei Provincial Clinical Research Center for the Diagnosis and Treatment of Pathogenic Fungal Infections. Standard strains were Candida parapsilosis ATCC22019, Aspergillus flavus ATCC204304, and Aspergillus flavus ATCC3375. All fungal strains were identified by microscopic morphology and molecular sequencing of internal transcribed spacer (ITS) ribosomal DNA and stored in 15% glycerol at -80°C. All sequences of all strains were uploaded to GenBank (accessions PP069948–PP070390). To accurately identify Aspergillus species, additional molecular sequence information for the β-tubulin and calmodulin genes was obtained. To ensure fungal spore viability and purity, all required strains were cultured on Sabouraud dextrose agar (SDA, Haibo Biological) at 35°C for 2–3 days before undergoing antifungal susceptibility testing.

[0027] 1.1.3 Culture medium

[0028] RPMI-1640 liquid culture medium: Take 10.4g of RPMI-1640 powder and 34.5g of MOPS, add 900mL of distilled water, mix thoroughly, adjust the pH to 7.0±0.1 with 1mol / L NaOH solution, bring the volume to 1000mL, filter with a 0.22μm diameter micromembrane for sterilization, and store at 4℃ for later use.

[0029] Sabouraud dextrose agar medium (SDA, Haibo Biological): 40g glucose; 10g peptone; 15g agar powder; 0.1g kanamycin. After autoclaving, aliquot the medium into petri dishes and store at 4°C for later use.

[0030] 1.1.4 Reagents and Instruments

[0031] DMSO (Beijing Bio-Innovation Technology Co., Ltd.), Agar Powder (Shanghai Yisheng Biotechnology Co., Ltd.), RPMI Medium (Roswell Park Memorial Institute) 1640 (Thermo Fisher Scientific (China) Co., Ltd.), NaOH (Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.), 3-(N-morpholino)propanesulfonic acid (MOPS, planktonic Acid) (Shanghai Yisheng Biotechnology Co., Ltd.), Vertical pressure steam sterilizer (Shanghai Shenan Medical Instrument Factory (LDZM-80KCS-Ⅲ)), Medical clean bench (Jinan Xinbeixi Biotechnology Co., Ltd. (BBS-SDC)), -20°C and 4°C refrigerators (Qingdao Haier Co., Ltd. (BCD-649WE)), 3 5°C biochemical incubator (Shanghai Lichen Instrument Technology Co., Ltd. (SPX-150BE)), electronic balance (Shanghai Huachao Industrial Co., Ltd. (HC311)); optical microscope (Shanghai Shangguang Industrial Co., Ltd. (E5-B)); pH adjuster (Shanghai Instrument & Electronics (Group) Co., Ltd. (PHS-25)); stainless steel spigot-type cup filter (Haining Delu New Material Technology Co., Ltd. (M-50)); pipette (Thermo Fisher Scientific (China) Co., Ltd.); hemocytometer (Shanghai Qiujing Biochemical Reagent Instrument Co., Ltd.); coverslips (Jiangsu Shitai Experimental Equipment Co., Ltd.).

[0032] 1.2 Experimental Methods

[0033] 1.2.1 Preparation of bacterial suspension

[0034] Fresh suspended conidia of Aspergillus, Candida, Epidermophyton dermatitidis, and Cryptococcus neoformans activated on SDA were collected using 0.9% physiological saline. Simultaneously, the concentrations of Aspergillus, Candida, Epidermophyton dermatitidis, and Cryptococcus neoformans were adjusted using a hematology analyzer to 3-5 × 10⁶ CFU / mL, 3-5 × 10⁵ CFU / mL, 3-5 × 10⁶ CFU / mL, and 3-5 × 10⁵ CFU / mL, respectively.

[0035] 1.2.2 Preparation and inoculation of antimicrobial susceptibility testing plates

[0036] In vitro antifungal susceptibility testing was performed using the checkerboard method. Following the microbroth dilution method (M27-A3), sterile 96-well plates were inoculated with *Candida glabrata* ATCC22019, *Aspergillus flavus* ATCC204304, and *Aspergillus flavus* ATCC3375 as control strains. In sterile 96-well plates, H1 served as a blank control. Wells A1-A9 were vertically inoculated with RPMI-1640 medium containing 50 µl of diluted antifungal agents (ITC, VRC, POS, FLU). The final working concentrations for ITC, VRC, and FLU ranged from 0.0625 to 8 μg / mL, and for POS, from 0.03125 to 4 μg / mL. Wells A1-A9 were horizontally inoculated with 50 µl of diluted GA, with a final working concentration of 1-64 μg / mL. The final concentrations of Aspergillus, Candida, Epidermophyton dermatitis, and Cryptococcus neoformans suspensions in RPMI-1640 medium were adjusted to 3-5 × 10⁴ CFU / mL, 3-5 × 10³ CFU / mL, 3-5 × 10⁴ CFU / mL, and 3-5 × 10³ CFU / mL, respectively. 100 µl of twice the final concentration of fungal suspension was added to each well of a 96-well plate. The plates were incubated at 35°C. Results for Candida and Cryptococcus neoformans were interpreted after 24 hours, results for Aspergillus after 48 hours, and results for Epidermophyton dermatitis after 60-72 hours.

[0037] 1.2.3 Result Interpretation

[0038] Colonies are identified visually, and the concentration that inhibits colony growth is taken as the minimum inhibitory concentration (MIC). The MIC values ​​are calculated, including those for each drug in both single-drug and combination therapy. The fractional inhibitory concentration index (FICI) is calculated by dividing the combined MIC of two drugs by the individual MICs of each drug: FICI = (MIC A combined) / (MIC A single drug) + (MIC B combined) / (MIC B single drug). The interaction relationships between the two drugs are as follows: FICI ≤ 0.5 indicates synergistic effect; > 0.5 and ≤ 4 indicate no interaction; > 4 indicates antagonistic effect.

[0039] 1.2.4 Quality Control

[0040] To ensure the accuracy of experimental results and eliminate errors from instruments, reagents, and operators, *Candida glabrata* ATCC 22019 was used to perform quality control on all drugs and culture media used in the same batch of experiments, with results referenced to CLSI M60-Ed2. All drug susceptibility tests were repeated three times.

[0041] Combined drug trial results

[0042] 2.1 For specific results regarding the in vitro interactions of GA in combination with triazole drugs against Aspergillus spp., please refer to Tables 1.1-1.2 and the appendix to the product information leaflet. Figure 1 .

[0043]

[0044]

[0045] Tables 1.1-1.2 clearly show that GA, when used alone, had no antifungal effect against 56 strains of Aspergillus. GA-ITC and GA-POS showed synergistic or no interaction with Aspergillus species, while the GA-VRC combination showed no interaction with Aspergillus species. Further analysis of the synergistic pattern revealed different results for the GA / ITC and GA / POS combinations. (See the attached instruction manual.) Figure 1 It can be clearly concluded that the GA-POS combination exhibits the broadest and best synergistic effect, especially with high synergistic rates in *Aspergillus niger* (9 / 10, 90%), *Aspergillus flavus* (9 / 10, 90%), and *Aspergillus terreus* (5 / 5, 100%), and a good synergistic rate was observed in *Aspergillus fumigatus* (26 / 31, 83.87%). The GA-ITC combination showed synergistic effects only in *Aspergillus fumigatus* (3 / 31, 9.68%) (see the instruction manual appendix). Figure 1 ).

[0046] 2.2 In vitro interaction of GA in combination with triazole drugs on dermatitis-causing fungi. Experimental results are shown in Table 2 and the appendix to the product manual. Figure 2 ;

[0047]

[0048] Table 2 clearly shows that GA alone had no antifungal effect against 16 strains of *Epiphyta dermatitidis*. The GA-ITC and GA-POS combinations showed synergistic effects and no interaction against *Epiphyta dermatitidis*, while the GA-VRC combination showed no interaction against *Epiphyta dermatitidis* (see Table 2). Figure 2 It can be concluded that no antagonistic effect was observed in the combination of GA and triazole drugs against *Tetranychus dermatitis*. The results indicate that the GA-POS combination showed the highest synergistic effect, and the combination of GA and triazole drugs demonstrated good safety and antibacterial efficacy against *Tetranychus dermatitis* (see [link to study]). Figure 2 ).

[0049] 3. In vitro interactions of GA combined with azole drugs against Candida spp. are shown in Tables 3.1-3.2 and the appendix to the product manual. Figure 3 ;

[0050]

[0051]

[0052] Tables 3.1-3.2 show that when used alone, GA showed no antifungal activity against 26 Candida strains. The GA-ITC and GA-POS combinations exhibited synergistic effects against Candida, with no interaction between them. The GA-VRC and GA-FLU combinations showed antagonistic effects against Candida, with no interaction between them. (See Tables 3.1-3.2)

[0053] Further analysis revealed varying synergistic and antagonistic effects among different strains. The synergistic effect of the GA-POS combination was primarily observed in *Candida auris* (8 / 10, 80%). The synergistic effect of the GA-ITC combination was observed in *Candida glabrata* (1 / 6, 16.67%), *Candida tropicalis* (1 / 3, 33.33%), and *Candida auris* (5 / 10, 50%). The GA-VRC combination showed varying degrees of antagonism in *Candida glabrata* (3 / 6, 50%), *Candida tropicalis* (2 / 3, 66.67%), and *Candida auris* (1 / 10, 10%). The GA-FLU combination showed antagonism in *Candida tropicalis* (2 / 3, 66.67%) and *Candida albicans* (2 / 6, 33.33%). Notably, *Candida* was the strain with the most frequent antagonism observed in this study. These results indicate that the GA-POS combination is far superior to GA-ITC, GA-VRC, or GA-FLU without any risk of antagonism. Furthermore, the combination of GA with VRC or FLU carries a clear clinical risk of antagonism, suggesting that the effects of GA combined with azoles on Candida are complex and species-dependent. The GA-VRC or GA-FLU combination should be used with caution for Candida tropicalis, and the GA-FLU combination should be avoided for Candida albicans. For Candida auris and Candida glabrata, caution should be exercised regarding the potential antagonistic effects of GA-VRC (see [link to relevant documentation]). Figure 3 ).

[0054] 4. In vitro interactions of GA combined with azole drugs against Cryptococcus neoformans; experimental results are shown in Table 4 and the appendix to the product manual. Figure 4 .

[0055]

[0056] From Table 4 and Figure 4 It can be concluded that when used alone, GA has no antifungal effect against the nine strains of Cryptococcus neoformans. The GA-ITC and GA-POS combinations showed synergistic effects and no interaction against Cryptococcus neoformans (see Table 4). The GA-VRC combination showed no interaction against Cryptococcus neoformans. The GA-FLU combination showed synergistic effects, no interaction, and antagonistic effects against Cryptococcus neoformans (see Table 4). Figure 4 ).

[0057] This invention, through experiments, reveals that glycyrrhetinic acid, in combination with itraconazole, posaconazole, voriconazole, and fluconazole, exhibits a biphasic effect against Aspergillus, Candida, Epidermophyton floccosum, and Cryptococcus neoformans. In vitro experiments show that glycyrrhetinic acid alone has no antifungal activity against these fungi. On the one hand, the combination of glycyrrhetinic acid with itraconazole and posaconazole shows good synergistic effects against Aspergillus, Epidermophyton floccosum, Candida, and Cryptococcus neoformans, with no antagonistic effects observed, demonstrating broad-spectrum synergistic potential. On the other hand, when glycyrrhetinic acid is used in combination with voriconazole and fluconazole, antagonistic effects are observed in some Candida species and Cryptococcus neoformans. This finding suggests that combined antifungal therapy is not always beneficial and may even carry risks, providing a new approach to promoting precise combined antifungal therapy.

Claims

1. The application of an azole drug in the preparation of antifungal drugs, characterized in that: Application of glycyrrhetinic acid combined with azole drugs in the preparation of antifungal drugs; The azole drug is posaconazole; the antifungal drug is an anti-Aspergillus terrestris drug; The ratio of glycyrrhetinic acid and posaconazole in the preparation of the anti-Aspergillus terrestris drug is (2-16):(0.03125-0.0625).

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