Application of cyanidin chloride in combination with fluconazole in the preparation of drugs for resisting drug-resistant fungi

Abstract

The application discloses application of cyanidin chloride or a pharmaceutical salt thereof in preparation of a drug for resisting drug-resistant fungi. The fungi are selected from drug-resistant Candida albicans. The application also provides application of cyanidin chloride or a pharmaceutical salt thereof in preparation of a drug-resistant fungus resisting reversal agent. The cyanidin chloride or the pharmaceutical salt thereof can be combined with fluconazole, a drug for resisting fungi, to improve the sensitivity of drug-resistant bacteria to fluconazole, realize the reversal of drug resistance, and resist fungi in a synergic way. Therefore, the cyanidin chloride or the pharmaceutical salt thereof can be used as the drug-resistant fungus resisting reversal agent.

Description

Technical Field

[0001] The present invention belongs to the technical field of drugs, and specifically relates to the application of cyanidin chloride in combination with fluconazole in the preparation of drugs for treating drug-resistant fungi. Background Art

[0002] Mycosis, that is, a disease caused by fungi. As a class of eukaryotes widely existing in nature, fungi can infect different parts of the human body. From the perspective of clinical pathogenic conditions, mycosis can be divided into two major categories: superficial mycosis and deep mycosis. Many problems such as the abuse of antibiotics have caused a certain degree of damage to the normal symbiotic relationship between bacteria and fungi; organ transplantation surgeries are also increasingly carried out clinically, and the use of immunosuppressants during and after the surgery affects the normal immune function of the body, reducing the body's resistance to fungi. The above problems have led to an increasing and more serious incidence of fungal infections in deep organs. Candida albicans is the main cause of fungal infections. However, in recent years, with the extensive use of antifungal drugs, the drug resistance of fungi has gradually increased. Strains resistant to fluconazole usually show cross-resistance to other azole drugs, making it very difficult to select clinical drugs for the treatment of Candida albicans infections. Therefore, finding a drug resistance reversal agent for antifungal drugs, improving the sensitivity of fungi to drugs, and producing a synergistic antifungal effect with existing drugs is an important method to improve the therapeutic effect of current existing drugs. Cyanidin chloride is a natural product derived from plants. From the current research situation, there is no report on the antifungal effect of cyanidin chloride.

[0003] Cyanidin chloride, Chinese name: cyanidin chloride, foreign name: cyanidin chloride, molecular formula: C 15 H 11 ClO6, Chinese alias: cyanidin chloride, CAS: 528-58-5, uses: can be used as food pigment, cosmetic raw material, etc. The structure is as follows:

[0004]

[0005] Patent application CN115400118A discloses an application of chlorocyanidin, specifically as follows: This invention discloses the antiviral function of chlorocyanidin, which can be used as an anti-influenza drug for the treatment or prevention of influenza. This invention provides the application of chlorocyanidin in the preparation of at least one of the following: 1) anti-influenza virus drugs or related products; 2) products that inhibit the replication or expression synthesis of influenza virus nucleic acid or protein components; 3) drugs or related products for treating diseases caused by influenza virus; 4) anti-inflammatory drugs or related products. Through a series of cell infection experiments on influenza A virus H1N1, this invention confirms that chlorocyanidin can effectively inhibit the replication and protein synthesis of influenza virus and has anti-inflammatory effects, alleviating the inflammatory response caused by viral infection. Therefore, it can be used to treat and prevent influenza infection, providing a new option for anti-influenza drugs. Summary of the Invention

[0006] The purpose of this invention is to provide the application of chlorocyanidin in combination with fluconazole in the preparation of drugs against drug-resistant fungi.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides the use of chlorocyanidin or its pharmaceutical salt in the preparation of antifungal drug resistance reversal agents, antifungal drug potentiators, or antifungal drug sensitizers.

[0009] The antifungal drug is an azole antifungal drug.

[0010] The azole antifungal drugs are selected from fluconazole, itraconazole, and voriconazole.

[0011] The fungi are selected from Candida (such as Candida albicans, Candida tropicalis, Candida krusei, etc.), Aspergillus (such as Aspergillus fumigatus), Cryptococcus (such as Cryptococcus neoformans, etc.).

[0012] The medicinal salt is an acid addition salt formed by cyanidin chloride with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

[0013] In a second aspect, the present invention provides the use of chlorocyanidin or its pharmaceutical salt in combination with fluconazole in the preparation of a drug for treating drug-resistant fungi.

[0014] The weight ratio of chlorocyanidin to fluconazole is 1:99 to 99:1; more preferably 1:50 to 50:1, more preferably 1:10 to 10:1; and most preferably 1:2.

[0015] The fungi are selected from Candida (such as Candida albicans, Candida tropicalis, Candida krusei, etc.), Aspergillus (such as Aspergillus fumigatus), Cryptococcus (such as Cryptococcus neoformans, etc.).

[0016] The medicinal salt is an acid addition salt formed by cyanidin chloride with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

[0017] A third aspect of the present invention provides a drug combination formulation comprising cyanidin chloride or its pharmaceutical salt and fluconazole in combination.

[0018] The weight ratio of chlorocyanidin to fluconazole is 1:99 to 99:1; more preferably 1:50 to 50:1, more preferably 1:10 to 10:1; and most preferably 1:2.

[0019] The chlorocyanidin or its pharmaceutical salt of the present invention can be used in combination with the azole antifungal drug fluconazole to increase the sensitivity of drug-resistant bacteria to fluconazole, thereby reversing drug resistance and synergistically fighting fungi. Therefore, the chlorocyanidin or its pharmaceutical salt of the present invention can be used as an antifungal drug resistance reversal agent (synergist / sensitizer).

[0020] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0021] This invention utilizes a checkerboard dilution method in in vitro drug susceptibility testing to evaluate the synergistic effect of cyanidin chloride and fluconazole against drug-resistant Candida albicans 103. Cyanidin chloride exhibits a significant synergistic antibacterial effect against fluconazole-resistant Candida albicans 103. When 4 μg / ml cyanidin chloride is used in combination with fluconazole, the MIC of fluconazole can be reduced. 80 Reducing the concentration of fluconazole from >64 μg / ml to below 8 μg / ml lowers the dosage by at least 8 times, significantly increasing the susceptibility of resistant fungi to fluconazole. Therefore, chlorocyanidin can be used as a synergist to enhance the antibacterial spectrum of fluconazole. Detailed Implementation

[0022] 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.

[0023] Example 1

[0024] Experiment on the synergistic effect of cyanidin chloride and fluconazole against drug-resistant fungi

[0025] In vitro drug susceptibility testing using the checkerboard dilution method was conducted to test the synergistic effect of cyanidin chloride and fluconazole against drug-resistant fungi.

[0026] The bacterial strain used was clinically isolated drug-resistant Candida albicans 103. All experimental bacteria were activated by streaking on Sabouraud dextrose agar (SDA) and incubated at 30°C for 2 weeks. Single colonies were then picked and reactivated by streaking, and the resulting single colonies were placed on SDA slants and incubated at 30°C for 2 weeks before being stored at 4°C. The culture medium was RPMI 1640 liquid medium, and all pre-culture treatments were performed according to standard methods. Fluconazole injection was purchased from Pfizer Inc. (Dalian); dimethyl sulfoxide (DMSO) was purchased from Shanghai Chemical Reagent Co., Ltd., China National Pharmaceutical Group.

[0027] Instruments used: Multiskan MK3 ELISA reader (Labsystems, Finland); water-jacketed electric thermostatic incubator (Shanghai Yuejin Medical Instrument Factory); MJX intelligent bacterial enzyme incubator (Ningbo Jiangnan Instrument Factory); THZ-82A benchtop thermostatic shaker (Shanghai Yuejin Medical Instrument Factory); SW-CT-IF ultra-clean workbench (Suzhou Antai Air Technology Co., Ltd.); inverted microscope (Amersham Pharmacia); micropipette (Finnpette, Finland); 96-well cell culture plate (Nunclon, Denmark).

[0028] Experimental steps:

[0029] Preparation of fungal suspension: Before the experiment, a small amount of drug-resistant Candida albicans 103 was picked from SDA medium stored at 4℃ using an inoculation ring and inoculated into 1 ml of YEPD medium. The mixture was then incubated at 30℃ with shaking at 200 rpm for 16 hours to activate the fungus into the late exponential growth phase. This bacterial suspension was then transferred to 1 ml of YEPD medium and activated again using the above method. After 16 hours, the cells were counted using a hemocytometer, and the bacterial concentration was adjusted to 1×10⁻⁶ using RPMI 1640 medium. 3 -5×10 3 CFU / ml.

[0030] Preparation of drug susceptibility testing plates: Take a sterile 96-well plate. Add 100 μl of RPMI 1640 liquid medium to well 1 of each row as a blank control; add 100 μl of freshly prepared bacterial suspension to wells 3-12; add 160 μl of bacterial suspension and 40 μl of the test compound solution to well 2; well 12 contains no drug, only 100 μl of bacterial suspension as a positive growth control. Serial dilutions are performed in wells 2-11 to achieve final drug concentrations of 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / ml, respectively, with DMSO content below 1% in each well. A quality control plate is prepared simultaneously with each drug susceptibility testing plate (quality control bacteria: according to the NCCLS M27-A protocol, *Candida glabrata* ATCC18062 is used as the quality control bacteria, with the following MIC reference values: Fluconazole (FCZ): MIC 80 MIC values ​​are 0.25-1.0 μg / ml; AmB: MIC values ​​are 0.5-2.0 μg / ml. This strain is used as the reference strain in each experiment; only when its MIC is... 80 The test is considered accurate and reliable only when the values ​​fall within the above range. If the tested strains also grow well, the test is considered successful and the results acceptable. Each antimicrobial susceptibility plate is incubated at 30℃.

[0031] Selection of in vitro drug susceptibility testing method: For evaluating the in vitro activity of cyanidin chloroform combined with fluconazole against drug-resistant fungi, the checkerboard microdilution method was selected. The checkerboard microdilution method is an extension of in vitro drug susceptibility testing, where the two drugs are serially diluted twofold in each of the two wells of a 96-well plate along the vertical (A to H) and horizontal (2 to 11) directions of a two-dimensional checkerboard. For example, when cyanidin chloroform was used in combination with the antifungal drug fluconazole, the final concentrations of fluconazole were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 g / ml, and the final concentrations of cyanidin chloroform were 64, 32, 16, 8, 4, 2, and 1 g / ml.

[0032] Evaluation Criteria: The partial inhibitory concentration index (FICI) is a key parameter for evaluating the interaction between two drugs in combination therapy. The fractional inhibitory concentration (FIC) is the ratio of the minimum inhibitory concentration (MIC) required for combined inhibition of bacteria by each drug to the MIC of either drug when used alone. The FICI index is the sum of the FIC values ​​of both drugs. When the MIC value is higher than the limit of detection (LOD), twice the LOD value is used to calculate the FICI.

[0033] The literature (Dai, et al., Design, synthesis, and evaluation of caffeic acid amides as synergists to sensitize fluconazole-resistant Candida albicans to fluconazole, Bioorganic & Medicinal Chemistry Letters, 2015, Vol. 25, Issue 1, Pages 34 - 37) reported that when FICI ≤ 0.5, the interaction between the two drugs was determined to be a synergistic effect, and the smaller the FIC index, the stronger the synergistic effect; when 0.5 < FICI ≤ 1, the interaction between the two drugs was determined to be an additive effect; when 1 < FICI ≤ 4, it was an irrelevant effect; when FICI > 4, the two drugs produced an antagonistic effect. The present invention selects the M27eA3 and M38eA2 standards formulated by the standard American Society for Microbiology CLSI: when FICI ≤ 0.5, the interaction between the two drugs is determined to be a synergistic effect; when 0.5 < FICI ≤ 1, the interaction between the two drugs is determined to be an additive effect; when 1 < FICI ≤ 4, it is an irrelevant effect; when FICI > 4, the two drugs produce an antagonistic effect.

[0034] The test results are shown in Table 1:

[0035] Table 1

[0036]

[0037]

[0038] MIC of fluconazole alone 80 > 64 μg / ml, MIC of cyanidin chloride alone 80 > 64 μg / ml. When 4 μg / ml of cyanidin chloride is combined with fluconazole, the MIC80 of fluconazole is reduced to ≤ 8 μg / ml, and FICI ≤ 0.094, greatly reducing the MIC concentration of fluconazole and improving the sensitivity of fungi to the drug. The interaction between the two drugs is determined to be a synergistic effect.

[0039] Experimental conclusion: The checkerboard dilution method was used for in vitro drug susceptibility testing of the synergistic effect of cyanidin chloride against fluconazole-resistant Candida albicans. Cyanidin chloride has an obvious synergistic antibacterial effect on Candida albicans 103 strains resistant to fluconazole, greatly reducing the MIC of fluconazole, and can be used as a synergist to improve the antibacterial spectrum of fluconazole.

[0040] 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 chlorocyanidin or its pharmaceutical salt in the preparation of antifungal drug resistance reversal agents, antifungal drug potentiators, or antifungal drug sensitizers, characterized in that, The antifungal drug is an azole antifungal drug; The azole antifungal drug mentioned is fluconazole; The fungus in question is Candida.

2. The application of chlorocyanidin or its pharmaceutical salt in combination with fluconazole in the preparation of drugs against drug-resistant fungi, characterized in that, The fungus in question is Candida.

3. The application of chlorocyanidin or its pharmaceutical salt as described in claim 2 in conjunction with fluconazole in the preparation of drugs against drug-resistant fungi, characterized in that, The weight ratio of cyanidin chloride to fluconazole is 1:99~99:1.

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