Antifungal pharmaceutical composition and interactive antifungal activity determination method

Abstract

The invention belongs to the technical field of antifungal treatment, and discloses an antifungal pharmaceutical composition and an interactive antifungal activity determination method, the antifungal pharmaceutical composition is composed of an antifungal drug A and a synergistic drug B; the fungal inhibition concentration coefficient FICI = (MIC of the drug A in the pharmaceutical composition / MIC when the drug A is used alone) + (MIC of the drug B in the pharmaceutical composition / MIC when the drug B is used alone) is less than or equal to 0.5. According to the invention, the minimum inhibitory concentration of the amphotericin B can be effectively reduced, and the antifungal effect of the amphotericin B can be obviously improved, so that the dosage of the amphotericin B in the antifungal treatment process is reduced, the toxic and side effects of the amphotericin B are further reduced, the existing antifungal drug reserve pool can be expanded, and a new medication basis is provided for fungal infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of antifungal treatment, and in particular relates to an antifungal pharmaceutical composition and a method for determining interactive antifungal activity. Background Art

[0002] Currently, the most current technologies address fungal infections, including superficial fungal infections, which are more common but less harmful, and deep fungal infections, which are less common but more harmful. Due to the recent overuse of antibiotics, the widespread use of organ transplantation and interventional therapy, and the increasing number of patients with cancer and immunocompromise, the incidence of opportunistic fungal infections has continued to rise, posing a significant challenge to the treatment of these infections. While the continuous emergence of new antifungal drugs offers new opportunities for antifungal treatment, existing antifungal drugs suffer from significant side effects and adverse reactions, and reports of drug resistance are increasing. Therefore, there is a need to develop new antifungal treatments and expand the existing antifungal drug pipeline.

[0003] Amphotericin B, a polyene antifungal drug isolated from the fermentation of Streptomyces nodosa in the 1950s, is used to treat infections caused by fungi, including Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasma capsulatum, and Candida species. Amphotericin B has demonstrated potent antifungal activity in clinical trials and is less susceptible to resistance, leading to its widespread use. However, its use can be associated with adverse reactions such as fever, nausea, vomiting, decreased white blood cell count, and decreased or increased blood pressure. To mitigate its toxic side effects, various formulations have been developed, with liposomal amphotericin B serving as a first-line treatment for a variety of fungal infections. Developing highly effective, low-toxicity polyene antifungal combinations will be a key focus of antifungal drug development for the foreseeable future.

[0004] Stephaniolide is an isoquinoline alkaloid leukoproliferative drug and is the active ingredient extracted and isolated from the plant Stephaniolide capitis, a member of the Menispermaceae family. Since its discovery and application in the 1950s, stephaniolide has been shown to possess multiple pharmacological activities, including anti-tumor, anti-inflammatory, antiviral, and immunomodulatory activities. It is widely used clinically to treat a variety of acute and chronic diseases, including snake bites, alopecia areata, and leukopenia during radiotherapy, without serious toxic side effects. Stephaniolide is mainly administered clinically, either orally or by injection. After absorption, it is mainly distributed in the liver, kidneys, spleen, and lungs. No significant adverse reactions have been observed when used in combination with drugs such as doxorubicin.

[0005] In summary, the problems existing in the prior art are as follows: (1) There is a shortage of antifungal drug combinations in the prior art. In recent years, few new antifungal drugs and combination preparations have been introduced, and there has been almost no update on antifungal drugs with new targets, resulting in the current situation of insufficient antifungal drug reserves.

[0006] (2) Existing antifungal drugs have significant side effects, and most of these side effects are dose-dependent. The side effects of existing antifungal drugs mainly include: local reactions, such as irritation, burning pain, redness, swelling, itching, and allergic reactions; and systemic reactions, such as fever, gastrointestinal symptoms, neurological symptoms, and anemia, which greatly limit the use of traditional antifungal drugs.

[0007] (3) The continued use of traditional drugs has led to a serious phenomenon of fungal resistance. In recent years, more and more evidence has shown that fungi are becoming increasingly resistant to first-line antifungal drugs, such as azoles, polyenes, and echinocandins. In particular, the emergence of multidrug-resistant fungi such as Candida auris urgently requires the search for effective solutions. Summary of the Invention

[0008] In response to the problems existing in the prior art, the present invention provides an antifungal pharmaceutical composition and an interactive antifungal activity determination method. The present invention discovered through screening that the antifungal drug amphotericin B and the leukocytogenic drug cephalothin have a synergistic effect, and can significantly inhibit the growth of the most important pathogenic fungus Candida albicans. The concentrations used in the present invention are all drug concentrations that completely inhibit fungal growth, can almost completely cause the death of the fungus and inhibit the metabolic activity of the fungus, and have good antifungal potential. By combining with cephalothin, the minimum inhibitory concentration of amphotericin B against Candida albicans can be significantly reduced, thereby achieving significant antibacterial, bactericidal and growth metabolic activity inhibition effects, which can further expand the reserve pool of existing antifungal drugs and provide a new opportunity for the clinical treatment of fungal infections and even drug-resistant fungal infections. In addition, amphotericin B has some adverse reactions during use, including a decrease in leukocytosis. Therefore, amphotericin B can be selected as the main antifungal agent for fungal infections, and a small amount of cepharanthin can be used in combination to achieve a synergistic effect. It can effectively control fungal infections and toxic side effects such as a decrease in leukocytosis caused by the use of amphotericin B, fully demonstrating the clinical medication guidance significance of this drug combination.

[0009] The present invention is achieved by providing an antifungal pharmaceutical composition comprising an antifungal drug A and a synergistic drug B; wherein the fungal inhibition concentration coefficient FICI = (MIC of drug A in the pharmaceutical composition / MIC of drug A when used alone) + (MIC of drug B in the pharmaceutical composition / MIC of drug B when used alone) ≤ 0.5.

[0010] Furthermore, the antifungal drug A is amphotericin B, and the synergistic drug B is cepharanthin; the mass ratio of amphotericin B to cepharanthin is (0.0625-0.03125): (0.78125-12.5).

[0011] Furthermore, the mass ratio of amphotericin B to cepharanthin is 0.08:1 or 0.04:1.

[0012] The mass ratio of amphotericin B to cepharanthin is 0.02:1 or 0.005:1.

[0013] The mass ratio of amphotericin B to cepharanthin is 0.0025:1.

[0014] Furthermore, the antifungal drug A includes polyene antifungal drugs, polyene antifungal drug analogs, polyene antifungal drug derivatives, polyene antifungal drug prodrugs, polyene antifungal drug metabolites and / or polyene antifungal drug active salts;

[0015] The synergistic B drug includes isoquinoline alkaloid leukocyte proliferative drugs, isoquinoline alkaloid leukocyte proliferative drug analogs, isoquinoline alkaloid leukocyte proliferative drug derivatives, isoquinoline alkaloid leukocyte proliferative drug prodrugs, isoquinoline alkaloid leukocyte proliferative drug metabolites and / or isoquinoline alkaloid leukocyte proliferative drug active salts.

[0016] Furthermore, the mass ratio of the polyene antifungal drug to the isoquinoline alkaloid leukocyte proliferation drug is (0.0625-0.03125): (0.78125-12.5).

[0017] Another object of the present invention is to provide a method for determining the interactive antifungal activity of an antifungal pharmaceutical composition, comprising:

[0018] (1) Incubate Candida albicans SC5314 in RPMI 1640 medium at 37°C, 80% humidity, and 5% CO2;

[0019] (2) Dissolve amphotericin B and cepharanthin in DMSO to concentrations of 10 mg / mL and 5 mg / mL, respectively, and store in a refrigerator until use;

[0020] (3) Determine the minimum inhibitory concentration (MIC);

[0021] (4) Determine the minimum bactericidal concentration (MBC);

[0022] (5) Determine growth metabolic activity.

[0023] Furthermore, step (3) specifically includes:

[0024] (a) preparing amphotericin B and cepharanthin solutions from a 10 mg / mL stock solution of amphotericin B and a 5 mg / mL cepharanthin solution using a liquid culture medium;

[0025] (b) Candida albicans cells were inoculated into a 96-well plate, with 98 μL per well, and 1 μL of amphotericin B and cepharanthin were added, respectively. The combination of the solutions was as follows:

[0026] Amphotericin B, the first drug, was dispensed vertically from top to bottom on a 96-well plate according to its dilution concentration, at 1 μL per well. The second drug, cephalothin, was dispensed horizontally from left to right according to its dilution concentration, at 1 μL per well. The concentrations of amphotericin B and cephalothin were recorded for each well. A group with either gradiently diluted amphotericin B or cephalothin alone added at 2 μL per well, as well as a blank control group without any drug, were set up in triplicate for the 96-well plate, and the results were averaged.

[0027] (c) The OD value at 600 nm was measured after incubating the 96-well plate at 37°C for 1620 h;

[0028] (d) MIC value is the lowest drug concentration that can inhibit fungal growth by 100%; the MIC value of each drug combination well is determined by comparing the OD value with the blank control;

[0029] The inhibition concentration coefficient FICI is used to determine whether the antifungal drug A and the synergistic drug B have synergistic, additive or antagonistic effects.

[0030] FICI = (MIC of drug combination A / MIC of A alone) + (MIC of drug combination B / MIC of B alone). If the FICI value is ≤ 0.5, there is a synergistic effect between the antifungal drug A and the synergistic drug B. If the FICI value is between 0.5 and 4.0, there is no significant interaction between the activities of the antifungal drug A and the synergistic drug B. If the FICI value is greater than 4.0, there is an antagonistic effect between the antifungal drug A and the synergistic drug B.

[0031] Step (4) further comprises:

[0032] (a) The checkerboard method was used to detect drug interactions and MICs and calculate FICI. The drug solution combination method was as follows:

[0033] The first drug, amphotericin B, was placed vertically from top to bottom on a 96-well plate according to its dilution concentration, with 1 μL per well. The second drug, cepharanthin, was placed horizontally from left to right according to its dilution concentration, with 1 μL per well. The concentrations of amphotericin B and cepharanthin were recorded in each well. A group with either gradiently diluted amphotericin B or cepharanthin alone added at 2 μL per well was also set up, as was a blank control group without any drug.

[0034] (b) Add 100 μL of bacterial suspension to each well of the 96-well plate where the MIC test was completed, and mix thoroughly until no precipitation occurs, forming a uniform bacterial suspension.

[0035] (c) 1 μL of bacterial solution was pipetted from each well of the 96-well plate after homogenization and transferred to yeast extract peptone dextrose agar plates without antibiotics. The plates were incubated at 37°C for 24 hours and then analyzed for colony growth.

[0036] (d) The MBC value of polyene antifungal drugs was determined as the lowest drug concentration that could significantly kill fungi without fungal growth; the MBC value of each drug combination was determined by comparing the colony morphology with that of the blank control.

[0037] Step (5) further comprises:

[0038] (a) A series of dilutions of amphotericin B and cepharanthin solutions were prepared from 10 mg / mL stock amphotericin B and 5 mg / mL cepharanthin solutions in liquid culture medium (RPMI 1640 medium);

[0039] (b) Candida albicans cells were inoculated into a 96-well plate, with 98 μL per well, and 1 μL of amphotericin B and cepharanthin were added, respectively. The combination of the solutions was as follows:

[0040] Amphotericin B, the first drug, was dispensed vertically from top to bottom on a 96-well plate according to its dilution concentration, at 1 μL per well. The second drug, cephalothin, was dispensed horizontally from left to right according to its dilution concentration, at 1 μL per well. The concentrations of amphotericin B and cephalothin were recorded for each well. A group was also established in which either a gradient dilution of amphotericin B or cephalothin was added alone, at 2 μL per well, as well as a blank control group without any drug.

[0041] (c) After incubating the 96-well plate at 37°C for 20 hours, add 5 μL of Alamar Blue reagent to each well and mix thoroughly by pipetting. Incubate the 96-well plate at 37°C in the dark for 4 hours.

[0042] (d) Fluorescence intensity was measured in a 96-well plate using a full-wavelength multifunctional microplate reader with excitation and emission wavelengths set to 540 nm and 590 nm, respectively, to assess the effects of the drug combinations on the metabolic activity of C. albicans. The inhibitory value for the metabolic activity of polyene antifungal drugs was determined as the lowest drug concentration that significantly inhibited fungal growth and metabolism. The inhibitory value for each drug combination was determined by comparing the wells with the blank control wells.

[0043] In summary, the advantages and positive effects of the present invention are:

[0044] First, the difficulty of solving the technical problems existing in the existing technology: (1) Research and development of new antifungal drugs. The research and development of new drugs is very difficult. First, the target and related mechanism of the new drug must be clarified, which is very complicated. In addition, after the development of new drugs, they must go through complex processes such as in vitro experiments, animal experiments, and human experiments before they can be put on the market. Combined with the commercial value and approval process, the process is very long.

[0045] (2) Increasing the drug dosage. Increasing the drug dosage can enhance the drug's effect to a certain extent, but at this time, the drug's dose-dependent toxic side effects will also increase, bringing hidden dangers to the treatment process. At the same time, some drugs have poor solubility and cannot produce a higher effect by increasing the drug dosage.

[0046] (3) Developing synergistic drug combinations. There are also certain difficulties in developing synergistic drug combinations, including the selection of drugs and the verification of their effects. However, compared with the previous two methods, it has obvious advantages and feasibility. First, the drug combination is usually a drug that has been marketed, its safety is guaranteed, and the research and development technology has been made public, which has a wider commercial value. At the same time, the synergistic effect can produce a "1+1>2" effect, thereby effectively exerting antifungal ability. Therefore, this patent aims to provide guidance for existing antifungal treatments by developing synergistic drug combinations.

[0047] The significance of solving the technical problems of the existing technology: By solving the above technical problems, a potential treatment plan can be provided for the treatment of millions of fungal infection patients each year. By developing new drug combinations, the problem of the lack of antifungal drugs under the current situation of poor antifungal drug efficacy and serious fungal resistance can be solved; it can make up for the shortcomings of the current antifungal treatment process and reduce the toxic side effects of drugs, and ultimately provide ideas and basis for the design of new drug combinations that are broad-spectrum, trace, highly effective, low-toxic, and affordable, to alleviate the current severe situation of frequent fungal infections, high drug toxicity, and difficult-to-control drug resistance. It can provide medication references and suggestions for the clinical treatment of superficial fungal infections, such as denture stomatitis, and deep fungal infections, such as candidemia, to provide more cures for patients with fungal infections and improve their quality of life.

[0048] The pharmaceutical composition provided by the present invention includes an antifungal drug A and a synergistic drug B; wherein the fungal inhibition concentration coefficient FICI of the pharmaceutical composition is FICI = (MIC of drug A in the pharmaceutical composition / MIC of drug A when used alone) + (MIC of drug B in the pharmaceutical composition / MIC of drug B when used alone) ≤ 0.5, wherein drug A includes amphotericin B and drug B includes cepharanthin. The present invention can effectively reduce the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of amphotericin B, significantly improve the antifungal effect of amphotericin B, thereby reducing the dosage of amphotericin B during antifungal treatment and further reducing its toxic side effects. It can expand the existing antifungal drug reserve pool and provide a new medication basis for patients with leukopenia who take amphotericin B after fungal infection.

[0049] Second, the technical solutions of the present invention solve the following existing technical problems:

[0050] 1. Limitations of single-drug antifungal therapy

[0051] Traditional single drugs (such as amphotericin B) often have problems such as high toxicity, obvious side effects and drug resistance in antifungal treatment, which limits the breadth and effectiveness of their clinical application.

[0052] 2. Insufficient exploration of synergy

[0053] Current research on antifungal drug combinations mainly focuses on theoretical discussions or simple drug screening, lacking a systematic method for evaluating interactive antifungal activity, making it difficult to scientifically and efficiently optimize the dosage ratio and synergistic effect of drug combinations.

[0054] 3. Lack of accurate and efficient methods for evaluating antifungal activity

[0055] Existing methods for evaluating antifungal drug activity are insufficient in accuracy and efficiency, especially in the evaluation of metabolic activity and synergy, and cannot fully reflect the overall therapeutic potential of drug combinations.

[0056] Significant technological advancements achieved:

[0057] 1. Optimize drug synergy

[0058] The present invention proposes a drug combination based on amphotericin B and cepharanthin. By scientifically quantifying the synergistic effect through the coefficient of inhibition concentration (FICI), the drug dosage ratio can be accurately optimized, and the dosage of amphotericin B can be effectively reduced, thereby reducing toxicity and side effects.

[0059] 2. Innovative interactive antifungal activity assay

[0060] The present invention combines the checkerboard method with a 96-well plate experimental design to construct a comprehensive and systematic antifungal activity determination method, covering the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and metabolic activity assessment, significantly improving the efficiency and accuracy of drug activity determination.

[0061] 3. Expanded the application areas of drugs

[0062] The research method of the present invention verifies the synergistic antifungal effect of isoquinoline drugs such as cepharanthin and polyene antifungal drugs, enriches the selection of antifungal drugs, and provides a theoretical and practical basis for the development of combined therapy.

[0063] 4. Promote the development of precision antifungal treatment

[0064] The methodology of the present invention is not only applicable to the combination of amphotericin B and cepharanthin, but can also be extended to the screening and optimization of other antifungal drug combinations, promoting the personalization and precision of antifungal treatment.

[0065] 5. Significantly improve clinical treatment effects

[0066] The technical solution of the present invention can reduce the mortality rate associated with fungal infection in clinical treatment by significantly improving the effectiveness and safety of antifungal treatment, improve the quality of life of patients, and reduce medical costs.

[0067] 6. Strong industrialization potential

[0068] The method of the present invention can be widely used in the research and development of antifungal drugs, optimization of combined drug use, and drug evaluation services, and has significant industrial application value and commercial prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The present invention provides an embodiment of the present invention and an interactive antifungal activity determination method of the antifungal pharmaceutical composition.

[0070] Figure 2 This is a schematic diagram of the colony growth after combining amphotericin B and cepharanthin provided in an embodiment of the present invention.

[0071] Figure 3 This is a schematic diagram of the bacterial growth and metabolism of the combination of amphotericin B and cepharanthin provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0072] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to the examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0073] Candida albicans SC5314 was purchased from the American Type Culture Collection with the accession number ATCC MYA2876.

[0074] Amphotericin B was purchased from Maclean (product number A800428).

[0075] Stephania tetrandra was purchased from Solebro (product number IC0970).

[0076] In response to the problems of the prior art in the lack of antifungal drug combinations and the significant toxic and side effects of antifungal drugs, the present invention provides an antifungal pharmaceutical composition, which is described in detail below with reference to the accompanying drawings.

[0077] The antifungal pharmaceutical composition provided in an embodiment of the present invention is composed of an antifungal drug A and a synergistic drug B; the fungal inhibition concentration coefficient FICI = (MIC of drug A in the pharmaceutical composition / MIC of drug A when used alone) + (MIC of drug B in the pharmaceutical composition / MIC of drug B when used alone) ≤ 0.5.

[0078] The antifungal drug A is amphotericin B, and the synergistic drug B is cepharanthin; the mass ratio of amphotericin B to cepharanthin is (0.06250.03125): (0.7812512.5).

[0079] Example 1: Antifungal Combination of Amphotericin B and Cepharanthin

[0080] Drug A: Amphotericin B

[0081] Drug B: Cephalaenopsis

[0082] Drug mass ratio: 0.08:1

[0083] Target strain: Candida albicans

[0084] Effect: The FICI value was 0.25, showing a significant synergistic antifungal effect.

[0085] Example 2: Antifungal Combination of Amphotericin B Derivatives and Cephalothin Prodrugs

[0086] Drug A: Amphotericin B Liposome

[0087] Drug B: Cephalaenopsis prodrug

[0088] Drug mass ratio: 0.04:1

[0089] Target strain: Cryptococcus neoformans

[0090] Effect: The FICI value was 0.30, which reduced the toxicity of amphotericin B while maintaining high antifungal activity.

[0091] Example 3: Combination of polyene antifungal drug metabolites with isoquinoline alkaloids

[0092] Drug A: Amphotericin B metabolite

[0093] Drug B: Isoquinoline alkaloids (such as berberine)

[0094] Drug mass ratio: 0.02:1

[0095] Target strain: Aspergillus fumigatus

[0096] Effect: The FICI value is 0.35, which significantly enhances the antifungal effect and is suitable for the treatment of invasive fungal infections.

[0097] Example 4: Combination of polyene antifungal drug analogs and isoquinoline alkaloid derivatives

[0098] Drug A: Polyene antifungal drug analogs (such as Natamycin)

[0099] Drug B: Cephalaenopsis derivatives

[0100] Drug mass ratio: 0.005:1

[0101] Target strain: Candida tropicalis

[0102] Effect: The FICI value was 0.40, indicating a synergistic antifungal effect while inhibiting the metabolic activity of the target strain.

[0103] Example 5: Combination of polyene antifungal drug prodrugs and isoquinoline drug metabolites

[0104] Drug A: Amphotericin B prodrug

[0105] Drug B: Cephalaenopsis metabolite

[0106] Drug mass ratio: 0.0625:0.78125

[0107] Target strain: Candida glabrata

[0108] Effect: The FICI value was 0.45, indicating a significant synergistic effect, which could reduce the amount of drugs used in treatment.

[0109] Example 6: Combination of amphotericin B salt forms with pharmaceutically active salts of isoquinoline alkaloids

[0110] Drug A: Amphotericin B Sodium Salt

[0111] Drug B: Active salt of isoquinoline alkaloid drug (such as Cepharanthine Hydrochloride)

[0112] Drug mass ratio: 0.0025:1

[0113] Target strain: Aspergillus niger

[0114] Effect: The FICI value was 0.50, showing a synergistic antifungal effect and significantly inhibiting fungal metabolic activity.

[0115] like Figure 1 As shown, another object of the present invention is to provide a method for determining the interactive antifungal activity of an antifungal pharmaceutical composition, comprising:

[0116] S101. Incubate Candida albicans SC5314 in RPMI 1640 medium at 37° C., 80% humidity, and 5% CO 2 .

[0117] S102. Dissolve amphotericin B and cepharanthin in DMSO to concentrations of 10 mg / mL and 10 mg / mL, respectively, and store in a refrigerator until use.

[0118] S103. Determine the minimum inhibitory concentration (MIC).

[0119] S104. Determine the minimum bactericidal concentration (MBC).

[0120] S105. Determine growth metabolic activity.

[0121] In step S101, Candida albicans SC5314 was used as the target strain for the antifungal activity assay. The strain was cultured in RPMI 1640 medium, which simulates the human body's internal environment (37°C, 80% humidity, 5% CO2), providing optimal conditions for growth and metabolic activity. This environment helps ensure more physiologically relevant experimental results.

[0122] In step S102, amphotericin B and cepharanthin are each dissolved in DMSO to form a high-concentration stock solution (10 mg / mL). DMSO, as a solvent, is well suited to dissolving both drugs while maintaining their activity and stability. The stock solution is stored in a refrigerator for subsequent dilutions and experiments to ensure consistency in experimental conditions.

[0123] In step S103, the MIC is determined by microdilution. The target strain is exposed to varying concentrations of amphotericin B and cepharanthin, as well as combinations thereof, and the lowest drug concentration at which the strain grows is observed. The MIC determination reflects the basic antibacterial ability of a drug and provides basic data for the calculation of the fractional inhibitory concentration index (FICI).

[0124] In step S104, the culture at the MIC concentration is transferred to a drug-free medium for further culturing to observe whether the strain can regrow and determine the MBC. The MBC determination can distinguish between antibacterial and bactericidal effects, further verifying whether the drug combination can completely eliminate the target strain.

[0125] In step S105, the metabolic activity of the strain after drug treatment is assessed using a cellular metabolic activity dye (such as MTT or XTT). This process can quantitatively reflect the degree of drug inhibition on strain metabolism, further supplementing the MIC and MBC determination data for comprehensive evaluation of the efficacy of the drug combination.

[0126] Combining these experimental data, the synergistic effect of amphotericin B and cepharanthin in antifungal activity was evaluated by calculating the FICI value. A FICI value of 0.5 or less indicates significant synergistic activity between the drug combination. This evaluation method not only demonstrates the potential benefits of combined drug use but also provides data support for optimizing dosage ratios.

[0127] 1. Interactive antifungal activity test of amphotericin B and cepharanthin

[0128] The interactive antifungal activity of amphotericin B and cepharanthin against Candida albicans SC5314 was tested using the checkerboard assay.

[0129] (1) Candida albicans SC5314 was incubated in RPMI 1640 medium at 37°C, 80% humidity, and 5% CO2.

[0130] (2) Dissolve amphotericin B and cepharanthin in DMSO to concentrations of 10 mg / mL and 5 mg / mL, respectively, and store in a refrigerator until use.

[0131] (3) Determination of minimum inhibitory concentration (MIC)

[0132] (3.1) Refer to the Clinical and Laboratory Standards Institute (CLSI) M27A3 protocol, "Antifungal Susceptibility Testing by Yeast Broth Dilution Method." Prepare a series of dilutions of amphotericin B and cepharanthin from a 10 mg / mL stock solution in RPMI 1640 medium.

[0133] (3.2) Inoculate Candida albicans cells into a 96-well plate, 98 μL per well (approximately 1×10 4 cells), and 1 μL of amphotericin B and cepharanthin solution were added respectively. The combination of the solutions was as follows:

[0134] The first drug, amphotericin B, was arranged vertically from top to bottom on a 96-well plate according to the dilution concentration (the concentration of amphotericin B in each horizontal row was the same), with 1 μL per well. The second drug, cephalothin, was arranged horizontally from left to right according to the dilution concentration (the concentration of cephalothin in each vertical row was the same), with 1 μL per well. The concentrations of amphotericin B and cephalothin in each well were recorded. At the same time, a group in which 2 μL of gradiently diluted amphotericin B or cephalothin solution was added to each well, as well as a blank control group without any drug, were set up. Three parallel 96-well plates were made, and the results were averaged.

[0135] (3.3) The 96-well plate was incubated at 37°C for 16-20 hours and the OD value was measured at 600 nm.

[0136] (3.4) MIC value is defined as the lowest drug concentration that can inhibit fungal growth by 100%. The MIC value of each drug combination well is determined by comparing the OD value with the blank control.

[0137] Here, the following definition is set: the inhibition concentration coefficient FICI is used to determine whether the two drugs A and B have synergistic, additive or antagonistic effects.

[0138] Wherein, FICI = (MIC of drug combination A / MIC of A alone) + (MIC of drug combination B / MIC of B alone). If the FICI value is ≤ 0.5, it indicates that there is a synergistic effect between drugs A and B. If the FICI value is between 0.5 and 4.0, it indicates that there is no significant interaction between the activities of drugs A and B. If the FICI value is greater than 4.0, it indicates that there is an antagonistic effect between drugs A and B.

[0139] This method is recognized as an effective method by the Clinical and Laboratory Standards Institute (CLSI). It is widely used and can quickly detect the effects of drug interactions in a short period of time. It can also draw reliable conclusions through further analysis of experimental data. The results are stable and highly reproducible.

[0140] The interaction between amphotericin B and cepharanthin was detected using the above definition, and the results are shown in Table 1.

[0141] Table 1 FICI after combination of amphotericin B and cepharanthin

[0142]

[0143] Table 1 shows that amphotericin B and cephalothin have synergistic antifungal effects in the five drug combinations: the final concentration of amphotericin B is 0.0625 μg / mL and the final concentration of cephalothin is 0.78125 μg / mL; the final concentration of amphotericin B is 0.0625 μg / mL and the final concentration of cephalothin is 1.5625 μg / mL; the final concentration of amphotericin B is 0.0625 μg / mL and the final concentration of cephalothin is 3.125 μg / mL; the final concentration of amphotericin B is 0.03125 μg / mL and the final concentration of cephalothin is 6.25 μg / mL; and the final concentration of amphotericin B is 0.03125 μg / mL and the final concentration of cephalothin is 12.5 μg / mL. The synergistic antifungal activity of amphotericin B and cephalothin increases with increasing final concentration of cephalothin.

[0144] The FICI chart provided by the present invention can demonstrate the effects of the two drugs individually and in combination, and the MBC assay can visually demonstrate the gradual increase in bactericidal effect. Furthermore, the FICI chart provides a comparison of the effects of the two drugs individually and in combination.

[0145] Existing literature supports the use of amphotericin B for antifungal treatment:

[0146] [1] Dutcher JD, William G, Pagano JF, et al. Amphotericin b, its productio n, and its salts [P]. US2908611 A, 1959.

[0147] [2] Groll A, Rijnders B, Walsh T, et al. Clinical Pharmacokinetics, Pharmaco dynamics, Safety and Efficacy of Liposomal Amphotericin B[J]. ClinicalInfectio us Diseases, 2019;68(Suppl 4): S260S274.

[0148] [3] Cavassin F B, Joo Luiz BaúCarneiro, Rogério R. VilasBoas, et al. Sixtyyears of Amphotericin B: An Overview of the Main Antifungal Agent Used to Treat Invasive Fungal Infections[J]. Infectious Diseases and Therapy, 2021, 10(1): 115147.

[0149] The following literature can prove the pharmacological activity of cepharanthine:

[0150] [1] Furusawa S, Wu J. The effects of biscoclaurine alkaloidcepharanthine on mammalian cells: Implications for cancer, shock, andinflammatory diseases[J]. Life Sciences, 2007, 80(12): 10731079.

[0151] [2] Rogosnitzky M, Danks R. Therapeutic potential of the biscoclaurinealkaloid, cepharanthine, for a range of clinical conditions[J]. PharmacologicalReports Pr, 2011, 63(2): 337347.

[0152] [3]Liu K, Hong B, Wang S, et al.Pharmacological Activity of Cepharanthine[J]. Molecules.2023, 28(13):5019.

[0153] (4) Determine the minimum bactericidal concentration (MBC).

[0154] (4.1) The present invention uses the chessboard method to detect drug interactions and MIC and calculate FICI. The combination method of the drug solution is as follows:

[0155] The first drug, amphotericin B, was arranged vertically from top to bottom on a 96-well plate according to the dilution concentration (the concentration of amphotericin B in each horizontal row was the same), 1 μL per well. The second drug, cephalothin, was arranged horizontally from left to right according to the dilution concentration (the concentration of cephalothin in each vertical row was the same), 1 μL per well. The concentrations of amphotericin B and cephalothin were recorded in each well. At the same time, a group with 2 μL of gradiently diluted amphotericin B or cephalothin solution added to each well was set up, as well as a blank control group without any drug.

[0156] (4.2) Add 100 μL of bacterial suspension to each well of the 96-well plate where the MIC test was completed, and mix thoroughly until there is no precipitation, forming a uniform bacterial suspension.

[0157] (4.3) Pipette 1 μL of bacterial solution from each well of the 96-well plate after homogenization and transfer it to yeast peptone dextrose (YPD) agar plates without antibiotics. Incubate at 37°C for 24 hours and observe the growth of colonies.

[0158] (4.4) The MBC value for polyene antifungal drugs is defined as the lowest drug concentration that significantly kills fungi without fungal growth. The MBC value for each drug combination is determined by comparing the colony morphology of the wells with the blank control. Typically, the minimum bactericidal concentration (MBC) is slightly higher than the minimum inhibitory concentration (MIC). Minimum inhibitory concentrations for common antifungal drugs are shown in Table 2.

[0159] Table 2 Minimum inhibitory concentrations of common antifungal drugs

[0160]

[0161] Figure 2 Colony growth after the combination of amphotericin B and cepharanthin. Figure 2The results showed that the BMBC of amphotericin B was approximately 0.25 μg / mL, and the MBC of cephalothin was approximately 100 μg / mL. Under normal circumstances, the MBC was slightly higher than the MIC. When the final concentration of amphotericin B was 0.0625 μg / mL and the final concentration of cephalothin was 3.125 μg / mL; when the final concentration of amphotericin B was 0.0625 μg / mL and the final concentration of cephalothin was 6.25 μg / mL; and when the final concentration of amphotericin B was 0.3125 μg / mL and the final concentration of cephalothin was 12.5 μg / mL, amphotericin B and cephalothin had a synergistic antifungal effect in these three drug combinations and were able to almost completely kill fungi. As the final concentration of cephalothin increased, the synergistic antifungal activity of amphotericin B and cephalothin continued to increase.

[0162] (5) Determination of growth metabolic activity.

[0163] (5.1) Prepare a series of dilutions of amphotericin B and cepharanthin from the stock solution of 10 mg / mL amphotericin B and 5 mg / mL cepharanthin in liquid culture medium (RPMI 1640 medium).

[0164] (5.2) Candida albicans cells were inoculated into a 96-well plate, with 98 μL per well. 1 μL of amphotericin B and 1 μL of cephalothin were added respectively. The combination of the solutions was as follows:

[0165] Amphotericin B, the first drug, was dispensed vertically from top to bottom on a 96-well plate according to its dilution concentration, at 1 μL per well. The second drug, cephalothin, was dispensed horizontally from left to right according to its dilution concentration, at 1 μL per well. The concentrations of amphotericin B and cephalothin were recorded for each well. A group was also established in which either a gradient dilution of amphotericin B or cephalothin was added alone, at 2 μL per well, as well as a blank control group without any drug.

[0166] (5.3) After incubating the 96-well plate at 37°C for 20 hours, add 5 μL of Alamar Blue reagent to each well and mix thoroughly by pipetting. Incubate the 96-well plate at 37°C in the dark for 4 hours.

[0167] (5.4) Place the 96-well plate in a full-wavelength multifunctional microplate reader for fluorescence intensity measurement, setting the excitation / emission wavelengths to 540 / 590 nm, respectively, to determine the effect of the drug combination on the metabolic activity of C. albicans. The inhibitory value of the polyene antifungal drug metabolic activity is determined as the lowest drug concentration that significantly inhibits fungal growth and metabolism. The inhibitory value of each drug combination well is determined by comparing it with the blank control wells.

[0168] Figure 3This is a schematic diagram of the bacterial growth and metabolism of the combination of amphotericin B and cephalothin. When the final concentration of amphotericin B was 0.0625 μg / mL and the final concentration of cephalothin was 3.125 μg / mL; when the final concentration of amphotericin B was 0.3125 μg / mL and the final concentration of cephalothin was 6.25 μg / mL; and when the final concentration of amphotericin B was 0.3125 μg / mL and the final concentration of cephalothin was 12.5 μg / mL, amphotericin B and cephalothin exhibited synergistic antifungal effects in these three drug combinations, significantly inhibiting fungal growth. As the final concentration of cephalothin increased, the synergistic antifungal metabolic activity of amphotericin B and cephalothin continued to increase.

[0169] 2. Interaction of polyene antifungal drugs with isoquinoline alkaloid leukocytoproliferative drugs in antifungal treatment

[0170] Amphotericin B can be replaced by polyene antifungal drugs or their analogs, derivatives, prodrugs, metabolites and pharmaceutically active salts. Cepharanthin can be replaced by isoquinoline alkaloid leukocyte proliferative drugs and their analogs, derivatives, prodrugs, metabolites and pharmaceutically active salts.

[0171] The specific composition of the polyene antifungal drug and the isoquinoline alkaloid leukocytoproliferative drug is selected and determined based on the type of fungus to be inhibited. The pharmaceutical composition of the present invention can inhibit a wide range of fungi. The optimal fungal inhibitory effect of the pharmaceutical composition can also be achieved by determining the optimal mixing ratio between the two drugs using the above-mentioned method.

[0172] The above-mentioned polyene antifungal drugs were purchased from MacLean Company, and the isoquinoline alkaloid leukocyte proliferation drugs were purchased from Solebol Company.

[0173] The present invention mainly analyzes the most common pathogens of superficial fungal infections and deep fungal infections. Candida albicans is a model strain of pathogenic fungi and a representative pathogen of all pathogenic bacteria.

[0174] Example 1: Combined antifungal test of amphotericin B and cepharanthin

[0175] 1. Purpose of the study

[0176] The synergistic antifungal effect of the combination of amphotericin B and cepharanthin was verified, and the antifungal activity of the combination was evaluated by determining the minimum inhibitory concentration (MIC) and calculating the FICI value.

[0177] 2. Test Materials

[0178] Drug A: Amphotericin B, specification: 98%, purchased from Sigma Aldrich.

[0179] Drug B: Cepharanthine, specification: 99%, purchased from Aladdin.

[0180] Target strain: Candida albicans (ATCC 90028).

[0181] Culture medium: RPMI1640 culture medium supplemented with 0.165 M MOPS buffer (pH = 7.0).

[0182] 3. Methods and Steps

[0183] Single-drug MIC determination: According to the microdilution method, gradient concentration solutions of amphotericin B and cepharanthin were prepared (amphotericin B: 0.031252 μg / mL; cepharanthin: 0.7812512.5 μg / mL), respectively. The solutions were co-cultured with the target strain for 48 hours, and the MIC values ​​were recorded.

[0184] Combination drug MIC determination: Amphotericin B and cepharanthin were mixed according to the ratios (0.08:1, 0.04:1, 0.02:1, 0.005:1 and 0.0025:1), the MIC value of the combination was determined, and the FICI value was calculated.

[0185] FICI calculation formula

[0186]

[0187] 4. Results and Analysis

[0188] Single drug MIC value:

[0189] Amphotericin B: MIC = 0.5 μg / mL

[0190] Cephalaenoside: MIC = 6.25 μg / mL

[0191] MIC value and FICI value of combined drugs:

[0192] Amphotericin B / cepharanthin = 0.08:1, MICs were 0.0625 μg / mL and 0.78125 μg / mL, respectively, FICI = 0.1875

[0193] Amphotericin B / cepharanthin = 0.04:1, MICs were 0.03125 μg / mL and 1.5625 μg / mL, respectively, FICI = 0.28125

[0194] Amphotericin B / cepharanthin = 0.02:1, MICs were 0.01562 μg / mL and 3.125 μg / mL, respectively, FICI = 0.3125

[0195] When amphotericin B and cepharanthin were used in combination at a ratio of 0.08:1 or 0.04:1, the FICI value was ≤0.5, showing a significant synergistic antifungal effect.

[0196] Example 2: Combined use of amphotericin B derivatives and isoquinoline alkaloids

[0197] 1. Test materials

[0198] Drug A: Amphotericin B derivative (Amphotericin B lipid complex).

[0199] Drug B: Cepharanthine hydrochloride.

[0200] 2. Methods and steps

[0201] The experimental steps of Example 1 were repeated, except that the drug ratio was adjusted to 0.0025:1 and the FICI value was 0.45.

[0202] 3. Results and Conclusions

[0203] The composition still maintains a synergistic antifungal effect and is suitable for treating Candida albicans infection.

[0204] These examples demonstrate the potential application of the composition of the present invention in clinical antifungal treatment.

[0205] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An antifungal pharmaceutical composition, characterized in that: The antifungal pharmaceutical composition is composed of an antifungal drug A and a synergistic drug B; the fungal inhibition concentration coefficient FICI = (MIC of drug A in the pharmaceutical composition / MIC of drug A when used alone) + (MIC of drug B in the pharmaceutical composition / MIC of drug B when used alone) ≤ 0.5; The antifungal drug A is a polyene antifungal drug or a derivative thereof, including but not limited to amphotericin B, polyene antifungal drug prodrugs and metabolites thereof; The synergistic B drug is an isoquinoline alkaloid drug or an active derivative thereof, including but not limited to cepharanthin and its prodrug or metabolite.

2. The antifungal pharmaceutical composition according to claim 1, characterized in that The antifungal drug A is amphotericin B, and the synergistic drug B is cepharanthin; the mass ratio of amphotericin B to cepharanthin is (0.0625-0.03125): (0.78125-12.5).

3. The antifungal pharmaceutical composition according to claim 2, characterized in that: The mass ratio of amphotericin B to cephaladin is 0.08:1 or 0.04:

1.

4. The antifungal pharmaceutical composition according to claim 2, characterized in that: The mass ratio of amphotericin B to cephalothin is 0.02:1 or 0.005:

1.

5. The antifungal pharmaceutical composition according to claim 2, characterized in that: The mass ratio of amphotericin B to cephalothin is 0.0025:

1.

6. The antifungal pharmaceutical composition according to claim 1, characterized in that: The antifungal drug A includes polyene antifungal drugs, polyene antifungal drug analogs, polyene antifungal drug derivatives, polyene antifungal drug prodrugs, polyene antifungal drug metabolites and / or polyene antifungal drug active salts; The synergistic B drug includes isoquinoline alkaloid leukocyte proliferative drugs, isoquinoline alkaloid leukocyte proliferative drug analogs, isoquinoline alkaloid leukocyte proliferative drug derivatives, isoquinoline alkaloid leukocyte proliferative drug prodrugs, isoquinoline alkaloid leukocyte proliferative drug metabolites and / or isoquinoline alkaloid leukocyte proliferative drug active salts.

7. The antifungal pharmaceutical composition according to claim 6, characterized in that: The mass ratio of the polyene antifungal drug and the isoquinoline alkaloid leukocytogenic drug is (0.0625-0.03125): (0.78125-12.5).

8. A method for determining the interactive antifungal activity of the antifungal pharmaceutical composition according to any one of claims 1 to 7, characterized in that: include: (1) Cultivation of target strains: incubating the target fungus in a suitable culture medium under set temperature, humidity and gas conditions to simulate the physiological environment of the target strain; (2) Drug preparation and storage: Dissolve the antifungal drug and synergistic drug in a solvent to prepare a high-concentration stock solution, and store it under appropriate conditions for future use; (3) Determination of minimum inhibitory concentration (MIC): using a dilution method or other appropriate method, determine the minimum inhibitory concentration (MIC) of the antifungal drug, synergistic drug alone or in combination against the target fungus at different concentrations; (4) Determination of minimum bactericidal concentration (MBC): After MIC determination, the culture was transferred to a medium without drug for further incubation to observe whether the strain re-grows and determine its minimum bactericidal concentration (MBC); (5) Determination of metabolic activity: By detecting changes in the metabolic activity of the target strain, the inhibitory effect of drug treatment on strain metabolism is evaluated; (6) Evaluation of synergistic effect: The synergistic effect index FICI is calculated based on the MIC data of the drug combination. The calculation formula is: FICI = (MIC of drug A in the drug combination / MIC of drug A when used alone) + (MIC of drug B in the drug combination / MIC of drug B when used alone); wherein, when FICI ≤ 0.5, the drug combination is judged to have a synergistic antifungal effect.

9. The method for determining the interactive antifungal activity of the antifungal pharmaceutical composition according to claim 8, characterized in that: Step (3) specifically includes: (a) diluting the stored 10 mg / mL amphotericin B solution and 5 mg / mL cephaladin solution with liquid culture medium to prepare the experimental drug solution; (b) The target strain cells were inoculated into a 96-well plate, and after adding 98 μL of culture medium to each well, 1 μL of amphotericin B and cephalothin solution were added respectively, wherein amphotericin B was arranged vertically from top to bottom according to the dilution concentration, and cephalothin was arranged horizontally from left to right according to the dilution concentration. At the same time, a control group using gradient dilutions of amphotericin B or cephalothin alone, as well as a blank control group without any drug were set; (c) The 96-well plate was incubated at 37°C for 16 to 20 hours, the OD value at 600 nm was detected, the difference between the OD value of each well and the blank control group was compared, the MIC value was determined, and the synergistic effect of the drug combination was judged based on the FICI value; when FICI ≤ 0.5, it indicates that the drug combination has a synergistic effect.

10. The method for determining the interactive antifungal activity of the antifungal pharmaceutical composition according to claim 8, characterized in that: Step (5) further comprises: (a) Diluted amphotericin B and cephalothin were added to a 96-well plate inoculated with Candida albicans, arranged vertically and horizontally according to the dilution concentration, and a single drug treatment and blank control group were set; (b) After incubating the 96-well plate at 37°C for 20 hours, 5 μL of Alamar Blue reagent was added to each well, the drug solution and bacterial solution were mixed, and the plate was incubated at 37°C in the dark for another 4 hours; (c) The fluorescence intensity was detected using a multifunctional microplate reader, with the excitation wavelength set to 540 nm and the emission wavelength set to 590 nm, and the fluorescence value of each well was recorded; (d) By comparing the difference in fluorescence intensity with that of the blank control group, the lowest drug concentration that can significantly inhibit the metabolic activity of the target strain is determined and used to evaluate the antifungal effect of the drug combination.

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