Application of crisaborole combined with azole antifungal drugs in the preparation of drugs against drug-resistant Candida albicans
The combined use of crisaborole and azole drugs solved the problem of increased drug resistance of Candida albicans, achieved effective inhibition of drug-resistant Candida albicans, and enhanced the antifungal treatment effect.
Patent Information
- Application Number
- CN202310719252.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-16
- Publication Date
- 2025-09-26
- Estimated Expiration
- 2043-06-16
AI Technical Summary
The increasing resistance of Candida albicans to existing azole antifungal drugs has led to difficulties in clinical infection control and the development of new drugs is time-consuming and labor-intensive.
Criborole is combined with azole antifungal drugs (such as fluconazole, voriconazole, and itraconazole) to prepare drugs against drug-resistant Candida albicans. The combined use of different drugs achieves synergistic effects and reduces the minimum inhibitory concentration.
The combination of crisaborole and azole drugs significantly reduced the minimum inhibitory concentration of drug-resistant Candida albicans, achieved effective inhibition of drug-resistant strains, and enhanced the antifungal treatment effect.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of medical technology, and particularly relates to the application of crisaborole combined with azole antifungal drugs in the preparation of drugs against drug-resistant Candida albicans. Background Art
[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.
[0003] Candida albicans is the most widespread opportunistic fungal pathogen in the human body, causing mucosal and systemic infections. Bloodstream infections caused by Candida species have a mortality rate of 40–60% in immunosuppressed and hospitalized patients. In addition to direct infection, Candida colonization and infection are also thought to be associated with pathophysiological conditions such as inflammatory bowel disease, cancer, diabetes, and metabolic diseases. Although invasive Candida infections caused by various non-albicans Candida species have increased, Candida albicans remains the most common pathogen causing invasive Candida infections.
[0004] Given that fungi, like their human hosts, are eukaryotic organisms, the number of unique molecular targets available for antifungal treatment is very limited. Currently, azoles remain one of the most commonly used antifungal drugs in clinical practice. However, long-term and widespread use has led to increased resistance to azoles, making clinical infection control more difficult. Given the low toxicity and widespread clinical application of azoles, as well as the time-consuming and labor-intensive nature of new drug development, combination therapy targeting common azole resistance pathways has become a hot topic in antifungal resistance research.
[0005] Crisaborole is a new nonsteroidal phosphodiesterase 4 (PDE4) inhibitor with the molecular formula C 14 H 12 BNO3. Developed by Anacor Pharmaceuticals (now acquired by Pfizer), crisaborole was approved by the U.S. FDA in 2016 for the treatment of mild to moderate atopic dermatitis in children two years of age and older. It is a topical ointment marketed as Staquis. It is the first new molecular entity approved by the FDA for the treatment of atopic dermatitis in the past 15 years. Summary of the Invention
[0006] In order to overcome the above problems, the present invention provides the use of crisaborole combined with azole antifungal drugs in the preparation of drugs against drug-resistant Candida albicans.
[0007] The first aspect of the present invention provides the use of crisaborole combined with azole antifungal drugs in the preparation of drugs against drug-resistant Candida albicans.
[0008] In one or more embodiments, the azole antifungal drug is one of fluconazole, voriconazole and itraconazole.
[0009] In one or more embodiments, the drug-resistant Candida albicans is: drug-resistant Candida albicans CA10, drug-resistant Candida albicans CA103, drug-resistant Candida albicans CA137 and drug-resistant Candida albicans CA632.
[0010] Preferably, when crisaborole is combined with fluconazole against drug-resistant Candida albicans CA10, the minimum inhibitory concentration of crisaborole is 8 μg / mL, and the minimum inhibitory concentration of fluconazole is 2 μg / mL;
[0011] When crisaborole was combined with fluconazole against drug-resistant Candida albicans CA103, the minimum inhibitory concentration of crisaborole was 4 μg / mL, and the minimum inhibitory concentration of fluconazole was 2 μg / mL;
[0012] When crisaborole was combined with fluconazole against drug-resistant Candida albicans CA137, the minimum inhibitory concentration of crisaborole was 8 μg / mL, and the minimum inhibitory concentration of fluconazole was 8 μg / mL;
[0013] When crisaborole was combined with fluconazole against drug-resistant Candida albicans CA632, the minimum inhibitory concentration of crisaborole was 16 μg / mL, and the minimum inhibitory concentration of fluconazole was 8 μg / mL.
[0014] Preferably, when crisaborole is combined with voriconazole against drug-resistant Candida albicans CA10, the minimum inhibitory concentration of crisaborole is 8 μg / mL, and the minimum inhibitory concentration of voriconazole is 0.0625 μg / mL;
[0015] When crisaborole was combined with voriconazole against drug-resistant Candida albicans CA103, the minimum inhibitory concentration of crisaborole was 4 μg / mL, and the minimum inhibitory concentration of voriconazole was 0.0625 μg / mL;
[0016] When crisaborole was combined with voriconazole against drug-resistant Candida albicans CA137, the minimum inhibitory concentration of crisaborole was 16 μg / mL, and the minimum inhibitory concentration of voriconazole was 0.0625 μg / mL;
[0017] When crisaborole was combined with voriconazole against drug-resistant Candida albicans CA632, the minimum inhibitory concentration of crisaborole was 16 μg / mL, and the minimum inhibitory concentration of voriconazole was 0.125 μg / mL.
[0018] Preferably, when crisaborole is combined with itraconazole against drug-resistant Candida albicans CA10, the minimum inhibitory concentration of crisaborole is 8 μg / mL, and the minimum inhibitory concentration of itraconazole is 0.125 μg / mL;
[0019] When crisaborole was combined with itraconazole against drug-resistant Candida albicans CA103, the minimum inhibitory concentration of crisaborole was 8 μg / mL, and the minimum inhibitory concentration of itraconazole was 0.25 μg / mL;
[0020] When crisaborole was combined with itraconazole against drug-resistant Candida albicans CA137, the minimum inhibitory concentration of crisaborole was 16 μg / mL, and the minimum inhibitory concentration of itraconazole was 0.25 μg / mL;
[0021] When crisaborole was combined with itraconazole against drug-resistant Candida albicans CA632, the minimum inhibitory concentration of crisaborole was 16 μg / mL, and the minimum inhibitory concentration of itraconazole was 0.5 μg / mL.
[0022] The second aspect of the present invention provides a drug for treating drug-resistant Candida albicans, which comprises crisaborole and an azole antifungal drug.
[0023] In one or more embodiments, the azole antifungal drug is one of fluconazole, voriconazole and itraconazole.
[0024] In one or more embodiments, the drug-resistant Candida albicans is: drug-resistant Candida albicans CA10, drug-resistant Candida albicans CA103, drug-resistant Candida albicans CA137 and drug-resistant Candida albicans CA632.
[0025] The third aspect of the present invention provides a pharmaceutical preparation comprising the above-mentioned drug against drug-resistant Candida albicans and a medical carrier or excipient.
[0026] The pharmaceutical carrier or excipient includes one or more pharmaceutically or food-acceptable diluents, wetting agents, binders, disintegrants, lubricants, regulators and other excipients.
[0027] The dosage forms of the pharmaceutical preparations are tablets, pills, sprays, capsules, granules, oral liquids, powders, syrups, injections, sprays, suppositories, etc.
[0028] (1) For the drug-resistant Candida albicans CA10, CA103, CA137, and CA632 strains described in the present invention, when the minimum inhibitory concentration of fluconazole alone was greater than 512 μg / mL, the minimum inhibitory concentration of crisaborole alone was 32-64 μg / mL, indicating that crisaborole alone had a certain inhibitory effect on these strains. When crisaborole was used in combination with fluconazole, the FICI values were all ≤0.5, indicating that fluconazole and crisaborole had a significant synergistic effect against drug-resistant Candida albicans.
[0029] (2) For the drug-resistant Candida albicans CA10, CA103, CA137, and CA632 strains described in the present invention, the minimum inhibitory concentration of voriconazole alone was 256-512 μg / mL, while the minimum inhibitory concentration of crisaborole alone was 32-64 μg / mL, indicating that crisaborole alone had a certain inhibitory effect on these strains. When crisaborole was used in combination with voriconazole, the FICI values were all ≤0.5, indicating that voriconazole and crisaborole had a significant synergistic effect against drug-resistant Candida albicans.
[0030] (3) For the drug-resistant strains of Candida albicans CA10, CA103, CA137, and CA632 described in the present invention, when the minimum inhibitory concentration of itraconazole alone was greater than 512 μg / mL, the minimum inhibitory concentration of crisaborole alone was 32-64 μg / mL, indicating that crisaborole alone had a certain inhibitory effect on these strains. When crisaborole was used in combination with fluconazole, the FICI values were all ≤0.5, indicating that fluconazole and crisaborole had a significant synergistic effect against drug-resistant Candida albicans. DETAILED DESCRIPTION
[0031] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.
[0032] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.
[0033] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.
[0034] Example 1
[0035] 1. The test materials and characterization methods used in the following examples are as follows:
[0036] Drug-resistant Candida albicans (CA10, CA103, CA137, CA632) were provided by Shandong University (identified);
[0037] The quality control strain was Candida albicans ATCC10231;
[0038] Yeast extract peptone glucose medium (Qingdao Haibo Biological Co., Ltd., batch number: 20220927);
[0039] RPMI1640 culture medium (Thermo Fisher Scientific, batch number: 2465381);
[0040] Crisaborole (Shanghai Yuanye Biotechnology Co., Ltd., batch number: S02GS159682);
[0041] Voriconazole (Shanghai Aladdin Biochemical Technology Co., Ltd., batch number: V129745);
[0042] Itraconazole (Shanghai Aladdin Biochemical Technology Co., Ltd., batch number: I129771);
[0043] Fluconazole (Dalian Meilun Biotechnology Co., Ltd., batch number: MB1288);
[0044] Electric constant temperature incubator (Shandong Olaibo Instrument Co., Ltd.);
[0045] Pipette (Eppendorf, Germany);
[0046] Microplate reader (Berteng, USA).
[0047] Preparation of bacterial suspension: Recover the fungal strain frozen at -80°C for 24 hours. After three consecutive recovery cycles, pick a single colony from the plate and inoculate it into yeast extract peptone glucose medium. Incubate at 35°C on a shaker for 16 hours. Dilute with sterile saline and adjust the concentration to 1×10 using a standard McFadden turbidimeter. 5 CFU / mL, and then the concentration of bacterial suspension was adjusted to 2×10 3 CFU / mL.
[0048] Preparation of drug stock solution:
[0049] Fluconazole stock solution: Place 20.48 mg of fluconazole powder in a sterile EP tube and add 1 mL of dimethyl sulfoxide to obtain a fluconazole stock solution with a concentration of 20480 μg / mL, which is Drug A.
[0050] Voriconazole stock solution: Place 20.48 mg of voriconazole in a sterile EP tube and add 1 mL of dimethyl sulfoxide to obtain a fluconazole stock solution with a concentration of 20480 μg / mL, which is Drug A B.
[0051] Itraconazole stock solution: Place 20.48 mg of itraconazole in a sterile EP tube and add 1 mL of dimethyl sulfoxide to obtain a fluconazole stock solution with a concentration of 20480 μg / mL, which is Drug A C.
[0052] Crisaborole stock solution: Place 25.6 mg of crisaborole powder in a sterile EP tube and add 2.5 mL of dimethyl sulfoxide to obtain a crisaborole stock solution with a concentration of 10240 μg / mL, which is Drug B.
[0053] 2. Drug Effect Determination
[0054] The following experimental procedures were performed in accordance with the regulations of the Clinical and Laboratory Standards Institute (M27-Ed4). All experiments were performed three times.
[0055] MIC of fluconazole, voriconazole and itraconazole alone: add 200 μL of bacterial solution (2×10 3 CFU / mL), and 100 μL of the same bacterial solution was added to each well in the remaining seven columns. The maximum drug concentration of 512 μg / mL was then added to each well in the first column. A two-fold dilution method was used: 100 μL was removed from the first well of each row and added to the second well, pipetting and mixing thoroughly. Another 100 μL was removed and added to the third well, mixing thoroughly. This was repeated until the penultimate well was thoroughly mixed, at which point 100 μL was removed and discarded. This resulted in a series of drug concentration gradients (512, 256, 128, 64, 32, 16, and 8 μg / mL), including a negative control. The sealed 96-well plate was placed in a 35°C incubator for 24 hours. The minimum inhibitory concentration (MIC) was recorded for fluconazole, voriconazole, and itraconazole alone, using the lowest concentration that resulted in no fungal growth as the minimum inhibitory concentration (MIC).
[0056] MIC of crisaborole alone: add 200 μL of bacterial solution (2×10 3 CFU / mL), and 100 μL of the same bacterial solution was added to each well of the remaining seven columns. Then, the maximum drug concentration of 512 μg / mL was added to each well of the first column. The two-fold dilution method was adopted, that is, 100 μL was taken from the first well of each row and added to the second well, pipetting and mixing, and then 100 μL was taken out and added to the third well to mix thoroughly, and so on until the second to last well was mixed and 100 μL was taken out and discarded, thus obtaining a series of drug concentration gradients (512, 256, 128, 64, 32, 16, 8 μg / mL) and including a negative control. The sealed 96-well plate was placed in a 35°C incubator for 24 hours, and the lowest concentration without fungal growth was taken as the minimum inhibitory concentration, and the MIC value of crisaborole alone was recorded.
[0057] MIC of fluconazole combined with crisaborole: 50 μL of fluconazole solution of different working concentrations (64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 μg / mL) was pipetted and added to the 2nd to 11th columns of a 96-well plate from left to right; 50 μL of crisaborole solution of different concentrations (64, 32, 16, 8, 4, 2, 1 μg / mL) was pipetted and added to rows G to A of a 96-well plate from bottom to top; after filling the 1st and H rows with culture medium to 100 μL, 2×10 3 Add 100 μL of bacterial solution containing 100 CFU / mL to each well in columns 1 through 11. Fill the well in column 12 with culture medium to 200 μL. Incubate the 96-well plate in a 35°C incubator for 24 hours. The minimum inhibitory concentration (MIC) is defined as the lowest concentration that results in no fungal growth. Select and record the MIC value of the two-drug combination for the optimal combination effect.
[0058] MIC of voriconazole combined with crisaborole: 50 μL of voriconazole solution of different working concentrations was pipetted from low to high concentrations (64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 μg / mL) and added to the 2nd to 11th columns of a 96-well plate from left to right; 50 μL of crisaborole solution of different concentrations was pipetted from low to high concentrations (64, 32, 16, 8, 4, 2, 1 μg / mL) and added to the G to A rows of a 96-well plate from bottom to top; after the 1st and H rows were filled to 100 μL with culture medium, 2×10 3 Add 100 μL of bacterial solution containing 100 CFU / mL to each well in columns 1 through 11. Fill column 12 to 200 μL with culture medium. Incubate the 96-well plate in a 35°C incubator for 24 hours. The minimum inhibitory concentration (MIC) is determined by determining the lowest concentration at which fungal growth is absent. The MIC of the two-drug combination for the optimal combination effect is then recorded.
[0059] MIC of the combination of itraconazole and crisaborole: 50 μL of itraconazole solution of different working concentrations was pipetted from low to high concentrations (64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 μg / mL) and added to the 2nd to 11th columns of a 96-well plate from left to right; 50 μL of crisaborole solution of different concentrations was pipetted from low to high concentrations (64, 32, 16, 8, 4, 2, 1 μg / mL) and added to the G to A rows of a 96-well plate from bottom to top; after the 1st and H rows were filled to 100 μL with culture medium, 2×10 3Add 100 μL of bacterial solution containing 100 CFU / mL to each well in columns 1 through 11. Fill column 12 to 200 μL with culture medium. Incubate the 96-well plate in a 35°C incubator for 24 hours. The minimum inhibitory concentration (MIC) is determined by determining the lowest concentration at which fungal growth is absent. The MIC of the two-drug combination for the optimal combination effect is then recorded.
[0060] 3. Synergistic Effect Evaluation
[0061] The combination of antimicrobial drugs in vitro or in vivo can show four effects: "independent", "additive", "synergistic" and "antagonistic". For the combination of different types of drugs, due to the different mechanisms and modes of action of the drugs, it is very likely to increase toxicity or induce the production of inactivated enzymes or cause antagonism due to competition for the same target; moreover, the same type of drugs does not necessarily have the same effects. For example, for cephalosporins, the first-generation cephalosporins are mainly anti-Gram-positive bacteria, while the third and fourth-generation cephalosporins are mainly anti-Gram-negative bacteria. In addition, only some third-generation and fourth-generation cephalosporins have anti-Pseudomonas aeruginosa effects. Therefore, for the combination of antimicrobial drugs, the antibacterial effect after combined use is extremely unpredictable.
[0062] The results of the combined drug sensitivity test use the following indicators to judge the interaction between the two drugs after combined use. FICI = MIC 甲药联用 / MIC 甲药单用 +MIC 乙药联用 / MIC 乙药单用 FICI≤0.5 indicates synergistic effect, 0.5<FICI≤1 indicates additive effect, FICI≤2 indicates irrelevant effect, and FICI>2 indicates antagonistic effect.
[0063] 4. Experimental results:
[0064] Table 1. Synergistic effect of crisaborole combined with fluconazole against drug-resistant Candida albicans
[0065]
[0066] Table 2. Synergistic effect of crisaborole combined with voriconazole against drug-resistant Candida albicans
[0067]
[0068] Table 3. Synergistic effect of crisaborole combined with itraconazole against drug-resistant Candida albicans
[0069]
[0070] For the present invention's anti-drug-resistant Candida albicans strains CA10, CA103, CA137, and CA632, when the minimum inhibitory concentration of fluconazole alone was greater than 512 μg / mL, the minimum inhibitory concentration of crisaborole alone was 32-64 μg / mL, indicating that crisaborole alone had a certain inhibitory effect on these strains. When crisaborole was used in combination with fluconazole, the FICI values were all ≤0.5, indicating that fluconazole and crisaborole had a significant synergistic effect when used together against drug-resistant Candida albicans.
[0071] For the drug-resistant Candida albicans CA10, CA103, CA137, and CA632 strains described in the present invention, the minimum inhibitory concentration of voriconazole alone was 256-512 μg / mL, while the minimum inhibitory concentration of crisaborole alone was 32-64 μg / mL, indicating that crisaborole alone had a certain inhibitory effect on these strains. When crisaborole was used in combination with voriconazole, the FICI values were all ≤0.5, indicating that voriconazole and crisaborole had a significant synergistic effect against drug-resistant Candida albicans.
[0072] For the drug-resistant Candida albicans CA10, CA103, CA137, and CA632 strains described in the present invention, when the minimum inhibitory concentration of itraconazole alone was greater than 512 μg / mL, the minimum inhibitory concentration of crisaborole alone was 32-64 μg / mL, indicating that crisaborole alone had a certain inhibitory effect on these strains. When crisaborole was used in combination with itraconazole, the FICI values were all ≤0.5, indicating that the combination of itraconazole and crisaborole had a significant synergistic effect against drug-resistant Candida albicans.
Claims
1. Application of crisaborole combined with azole antifungal drugs in the preparation of drugs against drug-resistant Candida albicans; The azole antifungal drug is one of fluconazole, voriconazole and itraconazole; The drug-resistant Candida albicans include: drug-resistant Candida albicans CA10, drug-resistant Candida albicans CA103, drug-resistant Candida albicans CA137 and drug-resistant Candida albicans CA632.
2. The use according to claim 1, characterized in that When crisaborole was combined with fluconazole against drug-resistant Candida albicans CA10, the minimum inhibitory concentration of crisaborole was 8 μg / mL, and the minimum inhibitory concentration of fluconazole was 2 μg / mL; When crisaborole was combined with fluconazole against drug-resistant Candida albicans CA103, the minimum inhibitory concentration of crisaborole was 4 μg / mL, and the minimum inhibitory concentration of fluconazole was 2 μg / mL; When crisaborole was combined with fluconazole against drug-resistant Candida albicans CA137, the minimum inhibitory concentration of crisaborole was 8 μg / mL, and the minimum inhibitory concentration of fluconazole was 8 μg / mL; When crisaborole was combined with fluconazole against drug-resistant Candida albicans CA632, the minimum inhibitory concentration of crisaborole was 16 μg / mL, and the minimum inhibitory concentration of fluconazole was 8 μg / mL.
3. The use according to claim 1, characterized in that When crisaborole was combined with voriconazole against drug-resistant Candida albicans CA10, the minimum inhibitory concentration of crisaborole was 8 μg / mL, and the minimum inhibitory concentration of voriconazole was 0.0625 μg / mL; When crisaborole was combined with voriconazole against drug-resistant Candida albicans CA103, the minimum inhibitory concentration of crisaborole was 4 μg / mL, and the minimum inhibitory concentration of voriconazole was 0.0625 μg / mL; When crisaborole was combined with voriconazole against drug-resistant Candida albicans CA137, the minimum inhibitory concentration of crisaborole was 16 μg / mL, and the minimum inhibitory concentration of voriconazole was 0.0625 μg / mL; When crisaborole was combined with voriconazole against drug-resistant Candida albicans CA632, the minimum inhibitory concentration of crisaborole was 16 μg / mL, and the minimum inhibitory concentration of voriconazole was 0.125 μg / mL.
4. The use according to claim 1, wherein When crisaborole was combined with itraconazole against drug-resistant Candida albicans CA10, the minimum inhibitory concentration of crisaborole was 8 μg / mL, and the minimum inhibitory concentration of itraconazole was 0.125 μg / mL; When crisaborole was combined with itraconazole against drug-resistant Candida albicans CA103, the minimum inhibitory concentration of crisaborole was 8 μg / mL, and the minimum inhibitory concentration of itraconazole was 0.25 μg / mL; When crisaborole was combined with itraconazole against drug-resistant Candida albicans CA137, the minimum inhibitory concentration of crisaborole was 16 μg / mL, and the minimum inhibitory concentration of itraconazole was 0.25 μg / mL; When crisaborole was combined with itraconazole against drug-resistant Candida albicans CA632, the minimum inhibitory concentration of crisaborole was 16 μg / mL, and the minimum inhibitory concentration of itraconazole was 0.5 μg / mL.
Citation Information
Patent Citations
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