Use of a pharmaceutical composition for the preparation of a medicament against Candida albicans

Abstract

The present application provides an anti-candida albicans pharmaceutical composition, which comprises Dp44mT and an antifungal drug; the antifungal drug is selected from one or more of azole antifungal drugs, polyene antifungal drugs, echinocandin antifungal drugs and flucytosine. The present application verifies that Dp44mT can enhance the anti-biofilm formation effect of common antifungal drugs, and enhance the mature biofilm destruction effect of common antifungal drugs, and further enhance the antifungal effect of common antifungal drugs, and reduce the formation of candida albicans drug resistance, thereby laying a theoretical foundation for a new treatment method for candidiasis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of a pharmaceutical composition in preparation of anti-candida albicans medicine. BACKGROUND

[0002] Candida albicans is a kind of fungus, which can cause superficial and deep infection, and even threaten life when the human body is in a low immunity state. It is estimated that more than 250,000 patients worldwide develop invasive candidiasis each year, and the mortality rate is more than 40%. Although more than 100 kinds of candida have been found, Candida albicans is still regarded as the most common conditional pathogenic species, 80% of candida infections are related to Candida albicans, and Candida albicans accounts for about 19% of infections in intensive care units. Therefore, Candida albicans infection often brings heavy burden to society and economy.

[0003] At present, the drugs for treating Candida albicans on the market mainly include azoles and polyene drugs, however, these drugs have defects such as narrow antibacterial spectrum, large toxicity and side effects to different extents; in addition, with the long-term and large-scale use of antifungal drugs, the drug resistance of Candida albicans is becoming more and more prominent. The drug resistance of Candida albicans may be related to the following reasons:

[0004] (1) Change of target molecule: change the structure of target protein to reduce drug sensitivity or overexpress target protein, thereby leading to drug failure, for example, mutation of ERG11 gene leads to change of amino acid sequence of azole drug target enzyme, thereby making azole drugs ineffective.

[0005] (2) Reduction of intracellular drug content: reduce intracellular drug content by reducing cell permeability or enhancing cell membrane efflux pump activity, for example, reduction of cytosine permease activity in Candida albicans cells can reduce the absorption of 5-FC, leading to drug resistance.

[0006] (3) Change of metabolic pathway: fungi can enhance drug resistance by changing metabolic pathways, for example, drug resistance to polyene compounds is mainly through functional deletion mutation of ergosterol biosynthesis gene to exhaust ergosterol, thereby producing alternative sterols, which cannot effectively interact with polyenes, thus reducing the sensitivity to antifungal drugs.

[0007] (4) Adaptive changes in cell wall: Fks1 protein is a catalytic subunit of glucosan synthetase complex, and mutation of the encoding gene may lead to reduced sensitivity to echinocandins.

[0008] (5) Formation of Candida albicans biofilm: Candida albicans biofilm can reduce the entry of drugs into cells, thereby increasing the drug resistance to commonly used antifungal drugs (such as fluconazole).

[0009] Although the mechanism of drug resistance to anti-Candida albicans drugs is known, there are few methods for enhancing antifungal effects or reversing drug resistance by affecting the drug resistance mechanism based on existing antifungal drugs. Therefore, exploring new small molecule compounds to enhance the antifungal effects of commonly used antifungal drugs to improve the treatment of Candida albicans infection is an effective means of anti-Candida albicans. SUMMARY

[0010] In order to overcome the defects in the prior art, the application provides a use of a pharmaceutical composition in the preparation of an anti-Candida albicans drug.

[0011] To achieve the above object, the application adopts the following technical solutions:

[0012] The first aspect of the application is to provide an anti-Candida albicans pharmaceutical composition, which comprises Dp44mT and an antifungal drug; the antifungal drug is selected from one or more of azole antifungal drugs, polyene antifungal drugs, echinocandin antifungal drugs and flucytosine.

[0013] Further, the above-mentioned azole antifungal drug is selected from one or more of fluconazole, voriconazole, posaconazole and itraconazole.

[0014] Further, the above-mentioned polyene antifungal drug is amphotericin B and / or nystatin.

[0015] Further, the above-mentioned echinocandin antifungal drug is selected from one or more of caspofungin, micafungin and anidulafungin.

[0016] The second aspect of the application is to provide the use of the above-mentioned pharmaceutical composition in the preparation of an anti-Candida albicans drug.

[0017] The third aspect of the application is to provide the use of the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating diseases caused by Candida albicans.

[0018] Further, the above-mentioned disease caused by Candida albicans is cutaneous candidiasis, mucosal candidiasis or internal organ and central nervous system candidiasis.

[0019] Further, the above-mentioned disease caused by Candida albicans is selected from skin inflammation, paronychia, perianal inflammation, inguinal inflammation, vaginitis, angular cheilitis, thrush, Candida albicans-induced pneumonia, gastroenteritis, endocarditis, meningitis and encephalitis.

[0020] Further, the above-mentioned drug is in the form of an external preparation, an injection or an oral preparation.

[0021] The application adopts the above technical solutions, and has the following technical effects compared with the prior art:

[0022] The application verifies that Dp44mT can enhance the anti-biofilm formation effect of common antifungal drugs, enhance the mature biofilm destruction effect of common antifungal drugs, further enhance the antifungal effect of common antifungal drugs, and reduce the formation of Candida albicans drug resistance, thereby laying a theoretical foundation for a new treatment method for candidiasis. DETAILED DESCRIPTION

[0023] The application will be described in detail and specifically below through specific examples, so that the application can be better understood. However, the following examples do not limit the scope of the application.

[0024] In the examples, the method is used unless otherwise specified, and the reagent is used unless otherwise specified.

[0025] Example 1

[0026] This example verifies that Dp44mT can enhance the anti-biofilm formation effect of common antifungal drugs, and the specific experimental steps and results are as follows:

[0027] 1. Biofilm formation chessboard experiment

[0028] After the activated Candida albicans is washed with PBS for three times, the concentration of the bacterial solution is diluted to 1×10 6 CFU / mL with RPMI1640 medium, and then 200 μL is plated in each well of a 96-well plate. Then the 96-well plate is placed in a 37℃ constant temperature incubator for culture for 90 min, which is recorded as the adhesion stage. After 90 min of culture, the 96-well plate is taken out, the supernatant is discarded, and the wells are washed with sterile PBS once, and then RPMI1640 medium without or with reagents is added, and the 96-well plate is placed in a 37℃ constant temperature incubator for culture for 24 hours, which is recorded as the proliferation stage.

[0029] Biofilm formation chessboard experiment: different concentrations of mixed reagents are added in the proliferation stage of biofilm formation.

[0030] ①Dp44mT and amphotericin B (Amphotericin B, AMP) chessboard experiment: the concentration of Dp44mT is 2 μg / mL to 0.008 μg / mL, and the concentration of AMP is 4 μg / mL to 0.0625 μg / mL.

[0031] ②Dp44mT and nystatin (Nystatin, NYS) chessboard experiment: the concentration of Dp44mT is 2 μg / mL to 0.008 μg / mL, and the concentration of NYS is 8 μg / mL to 0.125 μg / mL.

[0032] ③ Checkerboard test of Dp44mT and azoles: Dp44mT concentration ranged from 2 μg / mL to 0.03125 μg / mL, and azole concentration ranged from 64 μg / mL to 0.25 μg / mL.

[0033] 2. XTT Reduction Experiment

[0034] The detection of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazole-5-carboxanilide (XTT) is a colorimetric method that measures metabolic activity by measuring the reduction of the tetrazolium salt reagent XTT. Half an hour before the biofilm culture time reaches 24 hours, prepare fresh 0.5 mg / mL XTT and 0.32 mg / mL phenazinemethosulfate (PMS), vortex until homogeneous, then mix them in a 9:1 ratio, vortex again, and incubate in the dark. After the biofilm culture time reaches 24 hours, discard the culture medium, wash three times with PBS to remove the medium, and then add 100 μL of the XTT-PMS mixture to each well. Incubate at 37°C in the dark for 2 hours. After 2 hours, measure the OD. 450 The lowest reagent concentration at which the OD value becomes 20% or less of its original value is recorded as the minimum biofilm inhibitory concentration (MBIC).

[0035] 3. Calculation of Fractional Inhibitory Concentration Index (FICI)

[0036] The interaction between the two reagents was analyzed using the FICI model. The formula for calculating FICI is: FICI = FICIA + FICIB, where FICIA is calculated as MICA alone / MICA combined, and FICIB is calculated as MICB alone / MICB combined. MIC... (试剂单) =MBIC or MBEC, MIC when the reagent is used alone (试剂联) = MBIC or MBEC when reagents are used in combination. Among them, FICI ≤ 0.5 indicates synergistic effect; 0.5 < FICI ≤ 1 indicates additive effect; 1 < FICI < 4 indicates no interaction; FICI ≥ 4 indicates antagonistic effect.

[0037] The results are shown in Table 1 below, MBIC (AMP单) 2 μg / mL, MBIC (AMP联) 0.5 μg / mL, MBIC(Dp44mT单) 1 μg / mL, MBIC (Dp44mT联) The concentration was 0.008 μg / mL, and the FICI concentration was 0.258, indicating a synergistic effect. MBIC (NYS单) 8 μg / mL, MBIC (NYS联) 4 μg / mL, MBIC (Dp44mT单) 1 μg / mL, MBIC (Dp44mT联) With a concentration of 0.5 μg / mL and a FICI concentration of 1, they exhibit an additive effect. MBIC (唑类单) For values ​​greater than 64 μg / mL, MBIC (唑类联) 0.25 μg / mL, MBIC (Dp44mT单) 1 μg / mL, MBIC (Dp44mT联) The concentration is 0.5 μg / mL, and the FICI concentration is 0.503, therefore they have an additive interaction.

[0038] Table 1. Interactions between Dp44mT and antifungal drugs against Candida albicans biofilm formation.

[0039]

[0040]

[0041] Example 2

[0042] This embodiment verifies that Dp44mT can enhance the mature biofilm disruption effect of commonly used antifungal drugs. The specific experimental steps and results are as follows:

[0043] As described above, after Candida albicans forms a biofilm at 37°C for 24 hours, the culture medium is discarded, and the cells are washed three times with PBS. Prepared RPMI 1640 medium (with or without reagents) containing different concentrations of reagents is added to the wells containing the biofilm, and the cells are then incubated at 37°C for another 24 hours.

[0044] ① Checkerboard experiment of Dp44mT and AMP: Dp44mT concentration ranged from 256 μg / mL to 1 μg / mL, and AMP concentration ranged from 4 μg / mL to 0.0625 μg / mL.

[0045] ② Checkerboard experiment of Dp44mT and NYS: Dp44mT concentration ranged from 256 μg / mL to 1 μg / mL, and NYS concentration ranged from 64 μg / mL to 1 μg / mL.

[0046] The XTT reduction assay is used to determine the minimum biofilm eradication concentration (MBEC) required to disrupt the mature biofilm of Candida albicans, and then the fractional inhibition concentration index is calculated.

[0047] The results are shown in Table 2 below, MBEC (AMP单) 4 μg / mL, MBEC (AMP联) At 0.5 μg / mL, MBEC (Dp44mT单) Greater than 256 μg / mL, MBEC (Dp44mT联) The concentration was 128 μg / mL, and the FICI level was 0.375, therefore AMP and Dp44mT synergistically increased the disruption of mature biofilms. (MBEC) (NYS单) 8 μg / mL, MBEC (NYS联) 2 μg / mL, MBEC (Dp44mT单) Greater than 256 μg / mL, MBEC (Dp44mT联) The concentration was 128 μg / mL, and the FICI concentration was 0.5. Therefore, NYS and Dp44mT also have a synergistic effect in increasing the disruption of mature biofilms.

[0048] Table 2. Interactions between Dp44mT and antifungal drugs disrupting the mature biofilm of Candida albicans.

[0049]

[0050] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. The use of a pharmaceutical composition in the preparation of an anti-Candida albicans drug, characterized in that, The pharmaceutical composition comprises Dp44mT and amphotericin B.

2. The application according to claim 1, characterized in that, The drug may be in the form of a topical, injectable, or oral dosage form.

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