WO2023246487A1

WO2023246487A1 - Antifungal drug synergist

Info

Publication number: WO2023246487A1
Application number: PCT/CN2023/098301
Authority: WO
Inventors: 曹颖瑛; 朱臻宇; 刘志伟; 孙连娜; 黄豆豆; 朱全刚; 陈中建
Original assignee: 上海市皮肤病医院
Priority date: 2022-06-21
Filing date: 2023-06-05
Publication date: 2023-12-28
Also published as: CN115120602B; CN115120602A

Abstract

Provided is use of Thonningianin A in the preparation of an antifungal drug synergist. When combined with an antifungal drug, Thonningianin A not only can reduce the dosage of the drug, but also ensure the treatment effect on fungal infection, such that the antifungal drug recovers the effect on drug-resistant fungi. Therefore, Thonningianin A can be used as a synergist of an antifungal drug, which can reduce the dosage of antifungal drugs such as azoles, echinocandins or polyenes, thereby reducing the toxic and side effects of the drug, and effectively treating the fungal infection, especially drug-resistant fungal infection. Moreover, the effect of the antifungal drug on candida tropicalis and cryptococcus neoformans can be enhanced.

Description

An antifungal drug synergist

Technical field

The present invention relates to the field of medical technology, specifically, the application of Thonningianin A in the preparation of antifungal drug synergists.

Background technique

Invasive fungal infections have become one of the important causes of death in intensive care units (ICUs) in many countries in recent years. However, there are relatively few types of antifungal drugs available clinically, with high toxic and side effects, and clinically resistant strains are constantly isolated and reported, which makes the treatment of fungal infections face increasingly severe challenges. Antifungal drugs such as azoles, echinocandins, and polyenes are clinically commonly used antifungal drugs that can effectively treat deep and superficial fungal infections, such as fluconazole and voriconazole among azoles; echinocandins Caspofungin among bacteriocins; amphotericin B among polyenes, etc. However, as drug-resistant strains become more and more common in clinical settings, the dosage of drugs has to be increased, and some antifungal drugs are ineffective even with increased dosages. Increasing the dosage of medication will inevitably increase toxic side effects, especially drugs that have toxic side effects on the liver or heart, such as amphotericin B. In addition, the application of originally expensive drugs such as caspofungin and voriconazole will be limited.

Thonngianin A (THA) is a polyphenolic compound that widely exists in natural plants and has a series of biological activities. For example, THA can effectively inhibit rat liver glutathione S-transferase activity. In addition, THA can reduce the stress on oxidative human umbilical vein endothelial cells and aorta. THA can induce AMP/ATP-related AMPK activation by causing an increase in intracellular Ca2+ concentration, induce autophagy in HUVECs, reduce ROS levels in vitro, and inhibit oxidative stress-related oxidative stress in the aorta of atherosclerosis mouse models. expression of inflammasomes. However, there are no reports so far that THA can be used as a synergist for antifungal drugs such as azoles, echinocandins or polyenes.

Contents of the invention

The object of the present invention is to provide a new use of Thonningianin A (THA) in the preparation of antifungal drug synergists.

A first aspect of the present invention provides the use of Thonningianin A in the preparation of antifungal drug synergists. The structural formula of Thonningianin A is as shown in formula (I):

Experiments have shown that when antifungal drugs are combined with THA, THA can reduce the dosage of antifungal drugs, which not only ensures its antibacterial effect on fungi while reducing the dosage of antifungal drugs, but also reduces the resistance of clinical isolates. strains, Candida tropicalis or Cryptococcus neoformans all have significant synergistic effects. Therefore, THA can be used as a synergist with antifungal drugs for the treatment of different fungal infections.

Further, the antifungal drugs are azole, echinocandin and polyene antifungal drugs. Furthermore, the antifungal drugs are fluconazole, voriconazole, caspofungin, and amphotericin B.

Further, the fungi are Candida albicans, Candida tropicalis, and Cryptococcus neoformans.

A second aspect of the present invention provides the use of Thonningianin A in combination with antifungal drugs in the preparation of antifungal pharmaceutical compositions.

Further, the effective concentration of Thonningianin A in the antifungal pharmaceutical composition is 0.25-64 μg/ml.

A third aspect of the present invention provides an antifungal pharmaceutical composition, which is composed of Thonningianin A and antifungal drugs. The antifungal drugs are azole, echinocandin and polyene antifungal drugs.

Further, the antifungal drugs are fluconazole, voriconazole, caspofungin, and amphotericin B.

The advantages of the present invention are:

The present invention opens up a new use for THA. As a synergist for antifungal drugs, it can reduce the dosage of antifungal drugs such as azoles, echinocandins and polyenes, thereby reducing the toxic and side effects of the drugs. Especially fluconazole, voriconazole, caspofungin and amphotericin B. Especially THA as Synergists of antifungal drugs can restore the effect of antifungal drugs on drug-resistant fungi, effectively treat fungal infections, especially drug-resistant fungal infections, reduce the toxicity of drugs and reduce the economic burden of medication on patients. In addition, they can also enhance antifungal drugs. The drug's effect on drug-resistant strains, Candida tropicalis and Cryptococcus neoformans has important clinical application value.

Detailed ways

The specific implementation modes provided by the present invention will be described in detail below with reference to examples.

Example 1: Effect of combination of THA and fluconazole on different clinical fungal strains.

Materials and methods:

1. Reagent: THA and fluconazole are prepared into a mother solution with a concentration of 6.4 mg/ml using dimethyl sulfoxide, and stored at -20°C. Before the experiment, take out the drug storage solution and place it in a 35°C incubator to melt, mix thoroughly, and conduct a pharmacodynamic test.

2. Strains: Clinical strains of Candida albicans, Candida tropicalis and Cryptococcus neoformans were provided by the Mycology Laboratory of Shanghai Changhai Hospital, and were identified by morphology and biochemistry. All experimental strains were plated and activated on Sandcastle Dextrose Agar (SDA). After culturing at 35°C for 1 week, single clones were picked and plated again for activation. The single clones obtained for the second time were plated and activated on SDA slants. Method: After incubation, store at 4°C for later use.

3. Culture medium: RPMI 1640 Culture medium: RPMI 1640 (Gibco BRL Company) 10.0g, NaHCO ₃ 2.0g, morpholine propanesulfonic acid (Sigma) 34.5g, add 900ml of triple distilled water to dissolve, adjust the pH to 7.0 with 1N NaOH. Dilute to 1000ml, filter, sterilize, and store at 4°C. Sandcastle Dextrose Agar (SDA) medium: 10g peptone, 40g glucose, 18g agar, add 900ml distilled water to dissolve, add 50ml chloramphenicol aqueous solution with a concentration of 2mg/ml, adjust the pH to 7.0, dilute to 1000ml, and autoclave Store at 4°C after sterilization. YEPD culture medium: 10g yeast extract, 20g peptone, 20g glucose, add 900ml distilled water to dissolve, add 50ml chloramphenicol aqueous solution with a concentration of 2mg/ml, adjust the volume to 1000ml, and store at 4°C after autoclaving.

4. Instruments: Water-isolated electric heating constant temperature incubator (Shanghai Yuejin Medical Equipment Factory); THZ-82A desktop constant temperature oscillator (Shanghai Yuejin Medical Equipment Factory); Type 511 enzyme label analyzer (Shanghai Third Analytical Instrument Factory);

(1) Preparation of bacterial liquid: Candida bacterial liquid: Pick a small amount of Candida from the SDA medium stored at 4°C, inoculate it into 1ml YEPD culture medium, and activate it by shaking culture at 30°C at 200 rpm to keep the fungus in the late exponential growth phase. Take the bacterial liquid into 1ml YEPD culture medium, activate it again for 16 hours using the above method, count it with a hemocytometer, and adjust the bacterial liquid concentration to 3×10 ³ ~ 5 × 10 ³ with RPMI 1640 culture medium. pieces/ml.

(2) Cryptococcus neoformans liquid: Pick a small amount of Cryptococcus neoformans from the SDA medium stored at 4°C, inoculate it into 1ml YEPD culture medium, and activate it by shaking culture at 30°C at 200 rpm to make the fungus in the late exponential growth phase. Add the bacterial solution to 1 ml of YEPD culture medium, activate it again for 16 hours using the above method, count with a hemocytometer, and adjust the concentration of the bacterial liquid to 3×10 ³ to 5×10 ³ /ml using RPMI 1640 culture medium.

5. Preparation of drug susceptibility plates: Dilute the fluconazole and THA stock solutions with RPMI1640 culture medium to 64-0.0625 μg/ml. Take a sterile 96-well plate and add 100 μl of RPMI1640 culture medium containing or not containing a certain concentration of THA to wells 1 to 12 in each row; add corresponding culture medium and test drugs to well 2. Dilute the wells No. 2 to No. 11 twice, and then add 100 μl of bacterial solution to each row of wells No. 2 to No. 12, so that No. 1 is the RPMI 1640 culture medium without drugs as a negative control, and No. 12 is the culture medium without drugs. The bacterial liquid was used as a positive control. Each drug-sensitive plate was shaken and mixed on a shaker for 5 minutes and then incubated at 30°C.

6. Determination of MIC ₈₀ value: After culturing 96-well plates containing bacteria at 30°C for 48 hours, use an enzyme analyzer to measure the OD value of each well at 620 nm as usual. Compared with the positive control well, the drug concentration in the lowest concentration well where the OD value drops by more than 80% is MIC ₈₀ (the drug concentration at which fungal growth is 80% inhibited). When the MIC ₈₀ value of the drug exceeds the measured concentration range, statistics are made according to the following method: when the fluconazole MIC ₈₀ value is higher than the highest concentration of 64 μg/ml, it is counted as “>64 μg/ml”. The above experiments were performed in parallel 2 to 3 times. Only when the MIC ₈₀ value can be accurately repeated will it be accepted; when the MIC ₈₀ value differs by more than one concentration, the experiment needs to be repeated until the requirements are met.

The experimental results are shown in Table 1 and Table 2.

Table 1 MIC ₈₀ values of 5 clinical strains of Candida albicans when THA and fluconazole are used alone and in combination

Note: The units of fluconazole and THA are μg/ml (the same below).

Table 2 MIC ₈₀ values of 3 strains of Candida tropicalis and 3 strains of Cryptococcus neoformans when used alone and in combination with THA and fluconazole

It can be seen from Table 1 and Table 2 that when used together, THA can significantly reduce the MIC ₈₀ value of fluconazole for Candida albicans, Candida tropicalis and Cryptococcus neoformans, indicating that THA can enhance the antifungal effect of fluconazole.

Example 2: Effect of combination of THA and voriconazole on different clinical fungal strains.

Materials and methods

1. Reagent: THA and voriconazole are prepared into a mother solution with a concentration of 6.4 mg/ml using dimethyl sulfoxide, and stored at -20°C. Before the experiment, take out the drug storage solution and place it in a 35°C incubator to melt, mix thoroughly, and conduct a pharmacodynamic test.

2. Strains: Candida albicans, Candida tropicalis and Cryptococcus neoformans, provided by the Mycology Room of Shanghai Changhai Hospital and confirmed by morphological and biochemical identification.

Other experimental steps and methods are the same as in Example 1.

The experimental results are shown in Table 3 and Table 4.

Table 3 MIC ₈₀ values of 5 clinical strains of Candida albicans when THA and voriconazole were used alone and in combination

Note: The units of voriconazole and THA are μg/ml (the same below).

Table 4. MIC ₈₀ values of 3 strains of Candida tropicalis and 3 strains of Cryptococcus neoformans when used alone and in combination with THA and voriconazole.

It can be seen from Table 3 and Table 4 that when used together, THA can significantly reduce the MIC ₈₀ value of voriconazole by Candida albicans, Candida tropicalis and Cryptococcus neoformans, indicating that THA can enhance the antifungal effect of voriconazole.

Example 3: Effect of combined use of THA and caspofungin on different clinical fungal strains.

Materials and methods

1. Test drugs: THA is prepared into a mother solution with a concentration of 6.4 mg/ml in dimethyl sulfoxide, and caspofungin is prepared into a mother solution with a concentration of 0.2 mg/ml in dimethyl sulfoxide. The test drugs are stored at -20°C. Before the experiment, take out the drug storage solution and place it in a 35°C incubator to melt, mix thoroughly, and conduct pharmacodynamic tests separately.

Other experimental steps and methods are the same as in Example 1.

The experimental results are shown in Table 5 and Table 6.

Table 5 MIC ₈₀ values of 5 clinical strains of Candida albicans when THA and caspofungin are used alone and in combination

Note: The units of caspofungin and THA are μg/ml (the same below).

Table 6 MIC ₈₀ values of 3 strains of Candida tropicalis and 3 strains of Cryptococcus neoformans when used alone and in combination with THA and caspofungin

It can be seen from Table 5 and Table 6 that when used together, THA can significantly reduce the MIC ₈₀ value of caspofungin for Candida albicans, Candida tropicalis and Cryptococcus neoformans, indicating that THA can enhance the antifungal effect of caspofungin.

Example 4: Effect of combined use of THA and amphotericin B on different clinical fungal strains.

Materials and methods

1. Reagent: THA is prepared into a mother solution with a concentration of 6.4 mg/ml in dimethyl sulfoxide, and amphotericin B is prepared into a mother solution with a concentration of 2 mg/ml in dimethyl sulfoxide. The test drugs are stored at -20°C. Before the experiment, take out the drug storage solution and place it in a 35°C incubator to melt, mix thoroughly, and conduct pharmacodynamic tests separately.

Other experimental steps and methods are the same as in Example 1.

The experimental results are shown in Table 7 and Table 8.

Table 7 MIC ₈₀ values of 5 clinical strains of Candida albicans when THA and amphotericin B were used alone and in combination

Note: The units of caspofungin and THA are μg/ml (the same below).

Table 8 MIC ₈₀ values of 3 strains of Candida tropicalis and 3 strains of Cryptococcus neoformans when used alone and in combination with THA and amphotericin B

It can be seen from Table 7 and Table 8 that when used together, THA can significantly reduce the MIC ₈₀ value of Candida albicans, Candida tropicalis and Cryptococcus neoformans against amphotericin B, indicating that THA can enhance the antifungal effect of amphotericin B.

The preferred embodiments of the invention have been specifically described above, but the invention is not limited to the embodiments. Those skilled in the art can also make various equivalents without violating the spirit of the invention. modifications or substitutions, these equivalent modifications or substitutions are included in the scope defined by the claims of this application.

Claims

Application of Thonningianin A in the preparation of antifungal drug synergists. The structural formula of Thonningianin A is as shown in formula (I):
The application of ThonningianinA in the preparation of antifungal drug synergists according to claim 1, characterized in that the antifungal drugs are azole, echinocandin and polyene antifungal drugs.
The application of ThonningianinA in preparing antifungal drug synergists according to claim 2, characterized in that the antifungal drugs are fluconazole, voriconazole, caspofungin, and amphotericin B.
The application of ThonningianinA in the preparation of antifungal drug synergists according to claim 1, characterized in that the fungus is Candida albicans, Candida tropicalis, and Cryptococcus neoformans.
Application of ThonningianinA in combination with antifungal drugs in the preparation of antifungal pharmaceutical compositions.
The application of ThonningianinA in combination with an antifungal drug in preparing an antifungal pharmaceutical composition according to claim 5, wherein the effective concentration of ThonningianinA in the antifungal pharmaceutical composition is 0.25 to 64 μg/ml.
An antifungal pharmaceutical composition is characterized in that it consists of Thonningianin A and antifungal drugs, and the antifungal drugs are azole, echinocandin and polyene antifungal drugs.
The antifungal pharmaceutical composition according to claim 7, wherein the antifungal drug is fluconazole, voriconazole, caspofungin, or amphotericin B.
The antifungal pharmaceutical composition according to claim 8, wherein the fungus is Candida albicans, Candida tropicalis, or Cryptococcus neoformans.
The antifungal pharmaceutical composition according to claim 7, wherein the effective concentration of ThonningianinA in the antifungal pharmaceutical composition is 0.25-64 μg/ml.