A method for combining ambroxol and amphotericin B to fight Candida glabrata

Abstract

The invention discloses an aromatic plant extract used in combination with amphotericin B. The aromatic plant extract uses ambroxan as an active ingredient, opens up a new use for ambroxan, and serves as a synergist of antifungal drugs, thereby reducing the dosage of the antifungal drugs, thereby alleviating the toxic and side effects of the drugs, especially amphotericin B, and can effectively reduce the minimum inhibitory concentration (MIC) of amphotericin B; significantly improves 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 composition of ambroxan and amphotericin B in the preparation of an antifungal drug. Background Art

[0002] Fungal pathogens have a profound impact on global human health, food security, and biodiversity. Opportunistic fungal pathogens have become a leading cause of human mortality, especially in individuals with underlying health conditions or undergoing immunosuppressive therapy, with an annual mortality rate of approximately 1.5 million. The main pathogens include Candida, Aspergillus, and Cryptococcus. Globally, glabrata candidemia accounts for approximately 15% of all Candida-related systemic bloodstream infections. In some countries and regions, Candida glabrata has become the second most common Candida pathogen after Candida albicans. Infections caused by this species result in high morbidity and mortality (50%), and in bone marrow transplant patients, the mortality rate is as high as 100%.

[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]

[0009] Ambroxan belongs to the family of hemiaran diterpenes. It is reduced by clarylide and is a precursor of amino oxides. It is usually used as an important intermediate for the synthesis of ambergris, but it also has certain biological activity. Georgieva et al. found that ambroxan can reverse the hypokinesia caused by reserpine, and can enhance the hypokinesia caused by low-dose apomorphine, enhance mobility, and the effect of long-term treatment is more significant; ambroxan can increase the secretion of cortisol and ACTH; the study on the effect of ambroxan on convulsive epileptic seizures showed that the use of low-dose ambroxan has a protective effect on convulsions caused by sophora flavescens and bicuculline, and prolongs the latency period of convulsions.

[0010] In summary, although ambroxan has good biological activity, there has been no report on the use of ambroxan and polyene antifungal drugs as an antifungal drug composition. Therefore, the development of new antifungal drug compositions has great practical significance. Summary of the invention

[0011] One object of the present invention is to provide a new use of ambroxan as an antifungal pharmaceutical composition.

[0012] 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.

[0013] The invention provides an amphotericin B synergist. The synergist contains ambroxan as an active ingredient and may further include a pharmaceutically acceptable carrier.

[0014] The invention provides an antifungal composition, which comprises amphotericin B and ambroxan.

[0015] Preferably, the mass ratio of ambroxan to amphotericin B in the composition is (0.195-100):(0.125-0.5); preferably, the mass ratio of ambroxan to amphotericin B is 0.781:0.25; more preferably, the mass ratio of ambroxan to amphotericin B is (6.25-50):0.125.

[0016] The present invention also provides use of the composition in preparing antifungal drugs.

[0017] Preferably, the antifungal drug is an anti-Candida glabrata ATCC 2001 drug.

[0018] In addition, the antifungal pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.

[0019] Compared with the shortcomings and deficiencies of the prior art, the beneficial effects of the present invention are:

[0020] The invention discloses a method for synergistic antifungal treatment of a composition of ambroxan and amphotericin B, which provides a new use for ambroxan. 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

[0021] 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.

[0022] 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.

[0023] Ambroxan was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (product number: B21372).

[0024] Candida glabrata ATCC 2001 was purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0025] Amphotericin B was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Product No.: A105482).

[0026] Example 1 Antifungal activity test of the combination of ambroxan and amphotericin B.

[0027] The antifungal activity of ambroxan and amphotericin B against Candida glabrata ATCC 2001 was tested by the checkerboard dilution method.

[0028] 1. Inoculate Candida glabrata on YM plates and incubate at 28°C and 80% humidity for 48 hours.

[0029] 2. Dissolve ambroxol 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.

[0030] 3. Determination of minimum inhibitory concentration (MIC)

[0031] (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 ambrodiol and amphotericin B solutions from the stored 20 mg / ml ambrodiol and 0.8 mg / ml amphotericin B solutions.

[0032] (2) Candida glabrata ATCC 2001 was inoculated into a 96-well plate, with 100 μL (approximately 2 × 10 4 cells), 50 μL of ambroxan and amphotericin B were added respectively, and the combination of the solutions was as follows:

[0033] 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, ambroxol, was arranged horizontally from left to right according to the dilution concentration (all test wells in each column had the same ambroxol concentration), 50 μL per well. The concentrations of amphotericin B and ambroxol in each test group were recorded, and test wells containing different concentrations of ambroxol and amphotericin B were set up separately, with 100 μL added to each well, and a blank control group without any drug was set up, and three parallel 96-well plates were made, and the results were averaged.

[0034] (3) Incubate the 96-well plate at 37°C for 24 hours.

[0035] (4) The MIC value is defined as the lowest drug concentration at which the test wells are clear and no fungal growth is observed by visual observation.

[0036] Here, the following definition is set: the inhibition concentration coefficient FICI is used to determine whether the two drugs A and B have a synergistic, additive, 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 ambroxan and amphotericin B. Since ambroxan itself has poor anti-Candida glabrata activity, its (MIC 药物组合中的降龙涎二醇 / MIC 降龙涎二醇 ) can be ignored, and the results are shown in Table 1.

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

[0040]

[0041]

[0042] As can be seen from the above table, the final concentration of amphotericin B is 0.25μg / mL, and the final concentration of ambroxan is 0.781μg / mL; the final concentration of amphotericin B is 0.125μg / mL, and the final concentration of ambroxan is 6.25μg / mL; the final concentration of amphotericin B is 0.125μg / mL, and the final concentration of ambroxan is 12.5μg / mL; the final concentration of amphotericin B is 0.125μg / mL, and the final concentration of ambroxan is 25μg / mL; the final concentration of amphotericin B is 0.125μg / mL, and the final concentration of ambroxan is 50μg / mL. In a total of five groups of drug combinations, amphotericin B and ambroxan have synergistic antifungal effects. Among them, with the increase of ambroxan concentration, the synergistic anti-Candida glabrata activity of amphotericin B and ambroxan is continuously enhanced.

[0043] 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.

[0044] 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 ambroxan, the fungus is Candida glabrata ATCC2001, and the mass ratio of amphotericin B to ambroxan in the composition is 0.25:0.

781.

2. Use of a composition in the preparation of an antifungal drug, characterized in that: The composition consists of amphotericin B and ambroxan, the fungus is Candida glabrata ATCC2001, and the mass ratio of amphotericin B to ambroxan in the composition is 0.125:(6.25-50).

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