A method for combining sclareolide and amphotericin B to fight Cryptococcus neoformans

Abstract

The present invention discloses a method for combining sclareolide and amphotericin B to fight against new cryptococcus neoformans, and discloses an aromatic plant extract used in combination with amphotericin B. The aromatic plant extract uses sclareolide as an active ingredient, and opens up a new use for sclareolide. As a synergist of antifungal drugs, the dosage of the antifungal drugs can be reduced, thereby reducing the toxic and side effects of the drugs, especially amphotericin B, and can effectively reduce the minimum inhibitory concentration (MIC) of amphotericin B; significantly improve the antifungal effect of amphotericin B, thereby reducing the dosage of amphotericin B during treatment, and further reducing its toxic and side effects.

Description

Technical Field

[0001] The invention relates to the field of antifungal treatment, and is an application of a sclareolide and amphotericin B composition in the preparation of an antifungal drug. Background Art

[0002] Fungal pathogens have a profound impact on human health, food security, and biodiversity worldwide. Opportunistic fungal pathogens have become a leading cause of death in humans, particularly among individuals with underlying health conditions or receiving immunosuppressive therapy, with an estimated 1.5 million deaths per year. Major pathogens include Candida, Aspergillus, and Cryptococcus. Cryptococcus neoformans ( Cryptococcus neoformans ) is the most common conditional pathogenic fungus after Candida albicans. When the immune function is low or defective, systemic disseminated infection may occur, especially invading the central nervous system and causing cryptococcal meningitis (CNS). The mortality rate of cryptococcal meningitis (abbreviated as "cryptoencephalitis") is 10% to 30% in developed countries and 50% to 100% in developing countries with imperfect medical systems. In particular, the prevalence of acquired immunodeficiency syndrome (AIDS) has significantly increased the incidence of cryptococcal meningitis. In sub-Saharan Africa, cryptococcal meningitis has surpassed pneumococcal and meningococcal meningitis to become the most important community-acquired meningitis.

[0003] Microorganisms have a remarkable ability to develop resistance to antimicrobial drugs, threatening the efficacy of the limited antimicrobial drug arsenal and becoming a serious public health crisis. This is particularly worrisome for fungal pathogens, which can cause devastating invasive infections and for which only three classes of antifungal drugs are currently in clinical use, azoles, polyenes, and echinocandins, except for 5-fluorocytosine (5FC). Therefore, the emergence of resistance to these drugs by fungal pathogens has made fungal diseases a global human health problem.

[0004] A major obstacle to the discovery of antifungal drugs is the identification of important targets in fungi that can be specifically engaged by molecules lacking host toxicity. A promising strategy to expand the drug targeting space is to explore combination therapies. This approach, which is fundamental to the treatment of HIV, malaria, and tuberculosis, has multiple advantages, including reduced resistance rates, improved efficacy, reduced host toxicity, and expanded therapeutic scope. Drug combinations can also be used to target resistance mechanisms themselves, particularly those involved in resistance.

[0005] Robbins et al. screened six antifungal drugs at subinhibitory concentrations in combination with approximately 3,600 small molecules against four fungi. This study identified several small molecules, including FDA-approved drugs, that could improve the efficacy of antifungal drugs and even enhance the activity of antifungal drugs in resistant strains. Combinatorial screens can also be performed directly using resistant strains to identify compounds that can be used to treat resistant organisms and reduce the recognition of cross-resistant molecules. Screening using echinocandin-resistant Candida albicans showed that the metal chelator DTPA was an enhancer of caspofungin activity, and combination treatment with DPTA and caspofungin significantly improved the survival of mice inoculated with echinocandin-resistant strains compared to each drug alone.

[0006] High costs and severe side effects limit the use of combinations of antifungal drugs. In addition, contradictory results regarding synergistic or antagonistic effects of various antifungal combinations have also been reported. Therefore, the focus of research has shifted to examining combinations of typical antifungal drugs and non-antifungal drugs.

[0007] Amphotericin B (AmB) is a polyene antifungal drug with strong antifungal activity. Currently, amphotericin B is still the first choice for the treatment of many life-threatening deep fungal infections. Due to its broad antifungal spectrum, definite efficacy, and long half-life, it is known as the "gold standard" for the treatment of deep fungal infections. However, the defects of amphotericin B cannot be ignored. It is highly toxic and has many adverse reactions. Patients who receive ordinary injections often experience severe nephrotoxicity, hepatotoxicity, and hemolytic toxicity, accompanied by symptoms such as chills, chills, nausea, and vomiting. These serious side effects have severely limited the scope of clinical use of amphotericin B.

[0008] As a product of further chemical modification of sclareol, sclareolide is used in the synthesis of natural ambergris substitutes and the preparation of flavors. It also has good biological activity. In 2019, Khruengsai S et al. showed that sclareolide is an important antibacterial compound, which has certain antifungal effects on Trichoderma reesei and Diplodia theobromina. It can be used as a natural fungicide for plant diseases and used for the biocontrol of various agricultural plant pathogens. In 2020, Edouarzin Edruce et al. showed that drimane sesquiterpenes are a broad-spectrum antifungal compound. Sclareolide, as a type of drimane sesquiterpenes, can effectively kill Candida albicans, Cryptococcus, Aspergillus, Ascomycetes, Pneumocystis and other human pathogenic fungi. It is important to note that this terpenoid compound can also effectively eliminate fungi resistant to azole antifungal drugs such as fluconazole, such as Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida auris, etc. In the nematode model of Candida albicans infection, drimane can effectively eliminate Candida albicans in worms and has good tolerance. In 2020, Qing Chen et al. showed that clarysol is an EBOV entry inhibitor targeting the viral fusion process, and determined that clarysol has new biological activity against filamentous viruses.

[0009] In summary, although sclareolide has good biological activity, there has been no report on the use of sclareolide and polyene antifungal drugs as an antifungal drug combination. Therefore, the development of new antifungal drug combinations has great practical significance. Summary of the invention

[0010] The present invention aims at providing a new use of sclareolide as an antifungal pharmaceutical composition.

[0011] Another object of the present invention is to provide an antifungal composition, aiming to solve the problems of lack of antifungal drug combinations and large toxic and side effects of antifungal drugs in the prior art.

[0012] The invention provides an amphotericin B synergist. The synergist contains sclareolide as an active ingredient and may further include a pharmaceutically acceptable carrier.

[0013] The invention provides an antifungal composition, which comprises amphotericin B and sclareolide.

[0014] Preferably, the mass ratio of sclareolide to amphotericin B in the composition is (0.195-100):(0.125-0.5); preferably, the mass ratio of sclareolide to amphotericin B is (0.195-0.781):0.25; more preferably, the mass ratio of sclareolide to amphotericin B is (0.391-0.781):0.125.

[0015] The present invention also provides use of the composition in preparing antifungal drugs.

[0016] Preferably, the antifungal drug is an anti-Cryptococcus neoformans ( Cryptococcus neoformans ATCC32719) drug.

[0017] In addition, the antifungal pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.

[0018] Compared with the shortcomings and deficiencies of the prior art, the beneficial effects of the present invention are:

[0019] The invention discloses a method for synergistic antifungal effect of a composition of sclareolide and amphotericin B, which opens up a new use for sclareolide. As a synergist of antifungal drugs, the composition can effectively reduce the minimum inhibitory concentration (MIC) of amphotericin B, significantly improve the antifungal effect of amphotericin B, thereby reducing the dosage of amphotericin B during treatment, and further reducing its toxic side effects. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the invention more clear, the content of the invention is specifically described below through embodiments. It should be understood that the specific embodiments described here are only used to explain the invention and are not used to limit the invention.

[0021] It should be understood that 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.

[0022] Sclareolide was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (product number: B21373).

[0023] Cryptococcus neoformans Cryptococcus neoformans ATCC 32719 was purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0024] Amphotericin B was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (product number: A105482).

[0025] Example 1 Antifungal activity test of sclareolide and amphotericin B combined use.

[0026] Application of the checkerboard dilution method to the combination of sclareolide and amphotericin B against fungi (Cryptococcus neoformans Cryptococcus neoformans ATCC 32719) activity was tested.

[0027] 1. Inoculate Cryptococcus neoformans on YM plates and incubate at 26°C and 80% humidity for 72 hours.

[0028] 2. Dissolve sclareolide and amphotericin B in DMSO to a concentration of 20 mg / ml and 0.8 mg / ml respectively, and store in a -80°C refrigerator for later use.

[0029] 3. Determination of minimum inhibitory concentration (MIC)

[0030] (1) Refer to the Clinical and Laboratory Standards Institute (CLSI) version M27-A3, i.e., "Antifungal Susceptibility Test Protocol by Yeast Broth Dilution Method". Use the liquid culture medium (RPMI 1640) recommended in the protocol to prepare a series of dilution concentrations of sclareolide and amphotericin B solutions from the stored 20 mg / ml sclareolide and 0.8 mg / ml amphotericin B solutions.

[0031] (2) Cryptococcus neoformans ( Cryptococcus neoformans ATCC 32719) were inoculated into 96-well plates, with 100 μL per well (approximately 2 × 10 4 50 μL of sclareolide and amphotericin B were added to each cell. The combination of the solutions is as follows:

[0032] The first drug, amphotericin B, was arranged vertically from top to bottom on a 96-well plate according to the dilution concentration (all test wells in each horizontal row had the same amphotericin B concentration), 50 μL per well; the second drug, sclareolide, was arranged horizontally from left to right according to the dilution concentration (all test wells in each column had the same sclareolide concentration), 50 μL per well. The concentrations of amphotericin B and sclareolide in each test group were recorded, and test wells containing different concentrations of sclareolide and amphotericin B were set up separately, with 100 μL added to each well, as well as a blank control group without any drug, and three parallel 96-well plates were made, and the results were averaged.

[0033] (3) Incubate the 96-well plate at 37°C for 72 hours.

[0034] (4) The MIC value is defined as the lowest drug concentration at which the test wells are clear and no fungal growth is observed when visually observed.

[0035] Here, the following definition is used: The inhibition concentration coefficient FICI is used to determine whether two drugs A and B are synergistic or additive.

[0036] Or antagonistic effect.

[0037] Where, FICI=(MIC 药物组合中的A / MIC A单独A )+(MIC 药物组合中的B / MIC B单独B ), 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 the activities of drugs A and B are additive; if the FICI value is greater than 4.0, it indicates that there is an antagonistic effect between drugs A and B.

[0038] The above definition was used to detect the interaction between sclareolide and amphotericin B. Since sclareolide itself has poor anti-Cryptococcus neoformans activity, its (MIC 药物组合中的香紫苏内酯 / MIC 香紫苏内酯 ) can be ignored, and the results are shown in Table 1.

[0039] Table 1 FICI after combination of sclareolide and amphotericin B

[0040]

[0041] As can be seen from the above table, the final concentration of amphotericin B is 0.25μg / mL, and the final concentration of sclareolactone is 0.195μg / mL; the final concentration of amphotericin B is 0.25μg / mL, and the final concentration of sclareolactone is 0.391μg / mL; the final concentration of amphotericin B is 0.25μg / mL, and the final concentration of sclareolactone is 0.781μg / mL; the final concentration of amphotericin B is 0.125μg / mL, and the final concentration of sclareolactone is 0.391μg / mL; the final concentration of amphotericin B is 0.125μg / mL, and the final concentration of sclareolactone is 0.781μg / mL; the final concentration of amphotericin B is 0.125μg / mL, and the final concentration of sclareolactone is 1 .563μg / mL; the final concentration of amphotericin B was 0.125μg / mL, and the final concentration of sclareolactone was 3.125μg / mL; the final concentration of amphotericin B was 0.125μg / mL, and the final concentration of sclareolactone was 6.25μg / mL; the final concentration of amphotericin B was 0.125μg / mL, and the final concentration of sclareolactone was 12.5μg / mL; the final concentration of amphotericin B was 0.125μg / mL, and the final concentration of sclareolactone was 25μg / mL; the final concentration of amphotericin B was 0.125μg / mL, and the final concentration of sclareolactone was 50μg / mL. In a total of eleven groups of drug combinations, amphotericin B and sclareolactone had a synergistic antifungal effect. Among them, with the increase of sclareolactone concentration, the synergistic anti-neotypic activity of amphotericin B and sclareolactone against Cryptococcus neoformans continued to increase.

[0042] The fungi that can be inhibited by the pharmaceutical composition of the present invention come from a wide range of sources, including but not limited to shallow fungi that are less harmful to the body and deep fungi that are seriously harmful. The optimal inhibitory effect of the pharmaceutical composition on fungi can also be obtained by determining the optimal mixing ratio between the two through the above-mentioned implementation method.

[0043] 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 protection scope of the present invention.

Claims

1. Use of a composition in the preparation of an antifungal drug, characterized in that: The composition consists of amphotericin B and sclareolide, the fungus is Cryptococcus neoformans ATCC 32719, and the mass ratio of amphotericin B to sclareolide in the composition is 0.25:(0.195-0.781).

2. Use of a composition in the preparation of an antifungal drug, characterized in that: The composition consists of amphotericin B and sclareolide, the fungus is Cryptococcus neoformans ATCC 32719, and the mass ratio of amphotericin B to sclareolide in the composition is 0.125:(0.391-50).

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