Application of pharmaceutical composition in preparation of medicine for preventing and / or treating fungal infection

Abstract

The invention discloses application of a pharmaceutical composition in preparation of a medicine for preventing and / or treating fungal infection, and belongs to the technical field of medicines. The pharmaceutical composition comprises 6-O-methyl agrimony lactone or a pharmaceutically acceptable salt of the 6-O-methyl agrimony lactone, or a solvent complex and an azole antifungal drug. The 6-O-methyl hairyvein agrimony lactone and fluconazole have a synergistic effect after being combined, can resist invasion of candida albicans and drug-resistant strains thereof by protecting immune system functions, improve histopathologic injury of lungs and kidneys of infected persons, are fungal infection, and have the effect of preventing and treating the lung and kidney diseases. Especially, a new treatment strategy is provided for infection of Candida albicans and drug-resistant strains of the Candida albicans.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to application of a pharmaceutical composition in preparation of a medicine for preventing and / or treating fungal infection, and more particularly to application of 6-O-methyl ainslikiolide or a pharmaceutically acceptable salt or solvent complex thereof and an azole antifungal drug combination in preparation of a medicine for preventing and / or treating fungal infection. BACKGROUND

[0002] Invasive fungal infection has extremely strong infectivity and invasiveness, and among them, Candida albicans is the most common pathogen in fungal infection and has the highest mortality rate. About 70% of fungal infections in the world are caused by Candida albicans, and according to statistics, about 400,000 people in the world are infected with Candida albicans at different levels and sites every year, including superficial sites such as oral cavity, vagina, intestinal tract, etc. The severe cases (such as patients with low immunity) can further develop into invasive fungal infection, and even threaten the life safety of patients.

[0003] At present, there are mainly three types of drugs for treating invasive infection in clinical practice: polyenes, azoles and echinocandins. Among them, azole drugs are widely used in clinical practice due to their high safety and good efficacy. However, with the increase in the number of patients with malignant tumors and organ transplants, the long-term use of immunosuppressive agents and many other factors, the rate of invasive infection in clinical practice has shown an increasing trend year by year. In addition, in the process of long-term treatment and repeated drug use, fungal drug resistance is enhanced, and drug-resistant strains are continuously emerging in clinical practice. Drug-resistant Candida albicans infection is difficult to cure in clinical practice, and various complications are prone to occur, which brings heavy pressure to medical institutions and patient families. Therefore, the development of antifungal drugs has become an urgent need. SUMMARY

[0004] In view of the problems in the prior art, the present application provides application of a pharmaceutical composition in preparation of a medicine for preventing and / or treating fungal infection, wherein the pharmaceutical composition comprises 6-O-methyl ainslikiolide or a pharmaceutically acceptable salt or solvent complex thereof and an azole antifungal drug, and the medicine can resist the invasion of Candida albicans and its drug-resistant strains by protecting the function of the immune system, and improve the histopathological damage of the lung and kidney.

[0005] To achieve the object of the present application, the following technical solutions are adopted: Firstly, the present application provides application of a pharmaceutical composition in preparation of a medicine for preventing and / or treating fungal infection, wherein the pharmaceutical composition comprises 6-O-methyl ainslikiolide or a pharmaceutically acceptable salt or solvent complex thereof and an azole antifungal drug.

[0006] The 6-O-methyl ainslikiolide is a 6-hydroxymethyl derivative of ainslikiolide, and its chemical structural formula is shown in Figure 3 .

[0007] Preferably, the ratio of the 6-O-methyl aesculetin or its pharmaceutically acceptable salt, or solvent complex and the azole antifungal drug is 1-50:1, for example, it can be 1:1, 5:1, 10:1, 15:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, etc., preferably 13-26:1.

[0008] Preferably, the azole antifungal drug is fluconazole.

[0009] Preferably, the 6-O-methyl aesculetin is a racemate.

[0010] Preferably, the fungus is Candida albicans or drug-resistant Candida albicans.

[0011] Preferably, the drug can protect the immune system function against the invasion of drug-resistant Candida albicans.

[0012] Preferably, the form of the drug combination includes any one of the following forms: (1) 6-O-methyl aesculetin or its pharmaceutically acceptable salt, or solvent complex and azole antifungal drug are prepared into independent preparations, respectively; (2) 6-O-methyl aesculetin or its pharmaceutically acceptable salt, or solvent complex and azole antifungal drug are formulated into a compound preparation.

[0013] Preferably, the drug further includes a pharmaceutically acceptable pharmaceutical excipient.

[0014] Preferably, the pharmaceutically acceptable pharmaceutical excipient includes any one of or a combination of at least two of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant, a bacteriostatic agent, or a buffer.

[0015] Preferably, the drug dosage form is a tablet, a capsule, a granule, an injection, an oral liquid, a pill, a paste, a suspension, a dispersion, a syrup, a suppository, a gel, an aerosol, or a patch.

[0016] Compared with the prior art, the present application has the following beneficial effects: The present application unexpectedly found that the inhibitory activity of 6-O-methyl aesculetin combined with fluconazole on Candida albicans or drug-resistant Candida albicans is better than that of fluconazole, and the MIC 50The values are 6.5 μmol / L and 8 μmol / L, respectively, and the antifungal effect is obviously enhanced after 12 hours of combined administration; it is indicated that 6-O-methyl ainslixiol has a synergistic effect when combined with fluconazole, and can significantly improve the antifungal activity. Further animal studies show that combined administration can effectively alleviate the atrophy of the thymus and spleen of immunodeficient mice, improve the histopathological damage of the lung and kidney, and after the combination of 6-O-methyl ainslixiol and fluconazole, the immune system function can be protected to resist the invasion of Candida albicans and its drug-resistant strains, and the histopathological damage of the lung and kidney of the infected person is improved, which provides a new treatment strategy for fungal infection, especially Candida albicans and its drug-resistant strains. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The time-kill curve diagram of 6-O-methyl ainslixiol combined with fluconazole; Figure 2 The animal experiment result diagram of 6-O-methyl ainslixiol combined with fluconazole, wherein A is the mouse weight change diagram on the 1st day and the 8th day, B is the thymus organ coefficient of mice in each group, C is the spleen organ coefficient of mice in each group, D is the kidney HE staining and PASM staining sections of mice in each group after treatment, and E is the lung HE staining and PASM staining sections of mice in each group after treatment; Figure 3 The chemical structural formula of 6-O-methyl ainslixiol. DETAILED DESCRIPTION

[0018] The present application will be further described in detail below in combination with the drawings and examples, but in no way limits the present application, and any transformation or improvement based on the teaching of the present application falls within the protection scope of the present application.

[0019] The process, conditions, reagents, experimental methods, etc. for implementing the present application are all general knowledge and common sense in the art, and the present application has no special limitations. The experimental methods not specified in the examples are usually performed according to the conventional conditions or the conditions recommended by the manufacturers.

[0020] Unless otherwise specified, the meanings of all professional terms and scientific terms used in this specification are the same as those generally understood by the skilled person in the technical field to which the present application belongs. However, if there is a conflict, the present specification including the definition shall prevail.

[0021] The following are the sources of some reagents and consumables in the examples: 6-O-methyl ainslixiol is prepared in the laboratory of the inventors, and the preparation method is referred to (Zhang X, Mao ZW, Gao H. Synthesis of racemic ainslixiol [J]. Chemical Communications, 2024, 87(8): 988-991.). Fluconazole was obtained from Nanchang Hongyi Pharmaceutical Co., Ltd.; Candida albicans SC5314 was obtained from Kunming Medical University; Candida albicans ATCC14053-FR was obtained from Kunming Medical University; C57BL / 6 mice were obtained from Henan Sikebes Biotechnology Co., Ltd. Example 1 In vitro antifungal activity detection experiment.

[0022] 1. Minimum inhibitory concentration 50% (MIC 50 ) and Fractional Inhibitory Concentration Index (FICI) determination Drug solution preparation and dilution: 6-O-methylagrimone (I), fluconazole (FLC), a combination of fluconazole and 6-O-methylagrimone (FLC + I, 1:1 ratio), and a combination of fluconazole and agrimonone (FLC + AG, 1:1 ratio) were prepared to an initial stock solution of 200 µg / mL.

[0023] Dilutions in a 96-well plate (5-fold serial dilutions, 6 steps): Pre-add 80 μL of SDB to each well. Add 20 μL of a 1000 μg / mL solution to column 1 (highest concentration well) and mix thoroughly to obtain a concentration of 200 μg / mL. Pipette 20 μL from column 1 to column 2, mix thoroughly, and serially dilute to column 6, creating a six-step concentration gradient: 200, 40, 8, 1.6, 0.32, and 0.064 μg / mL. Column 7 is a blank control (SDB alone); column 8 is a growth control (SDB + bacterial solution). Set up three replicate wells for each concentration.

[0024] Preparation and inoculation of bacterial suspension: Activate the target strains, Candida albicans SC5314 and ATCC14053-FR, for two generations on SDB medium. Prepare the bacterial suspension using sterile saline or PBS, and adjust the concentration using a hemocytometer or McFarland turbidimeter. Add 100 µL of the suspension to each well of a 96-well plate (including the drug gradient wells, negative control wells, and positive control wells) where drug dilutions have been completed. The final initial inoculum concentration of Candida albicans in each well is 1 × 10 5 CFU / mL.

[0025] Culture: Place the inoculated 96-well plate in a 37°C constant temperature incubator and incubate for 24 hours.

[0026] Result determination and analysis: After incubation, the optical density (OD) of each well was measured at a wavelength of 625 nm using a microplate reader. MIC was calculated.50 :According to the OD value, the minimum inhibitory concentration (MIC) of each drug (single drug and combination) against Candida albicans was determined at 50%. 50 ), which is the lowest drug concentration that inhibits 50% fungal growth. Calculation of FICI: For the combination group, according to their respective MIC 50 The concentrations in the combined wells were used to calculate the Fractional Inhibitory Concentration Index (FICI) to assess the interaction between the two drugs (synergistic, additive, unrelated, or antagonistic). The results are shown in Table 1.

[0027] Table 1 Inhibitory effect of fluconazole combined with 6-O-methyl agrimony lactone on Candida albicans

[0028] As can be seen from Table 1, when methyl agrimonolide (I) of the present invention is used alone, it has no obvious inhibitory activity against the sensitive strain SC5314 of Candida albicans and the drug-resistant strain ATCC14053-FR of Candida albicans. However, when used in combination with fluconazole, it has a good inhibitory effect against both Candida albicans SC5314 and the drug-resistant Candida albicans ATCC14053-FR. 50 The values ​​were 6.5 μmol / L and 8 μmol / L, respectively, showing a good synergistic effect.

[0029] 2. Draw a time-kill curve for drug-resistant Candida albicans ATCC14053-FR Take the drug-resistant Candida albicans ATCC14053-FR in the logarithmic growth phase and adjust the bacterial solution concentration to 1×10 5 CFU / mL, 6-O-methyl agrimonolide (I), fluconazole (FLC) and a combination of fluconazole and 6-O-methyl agrimonolide (FLC+I) were diluted to a final concentration of 8 µmol / L, 1:1 mass ratio of FLC to I, and incubated in a shaker at 30°C, 150 rpm for 48 h. SDB alone and untreated Candida albicans served as blank and control cultures. After incubation for 0, 4, 8, 12, 24, 36, and 48 h, the solutions were thoroughly mixed at designated time points. 200 µL of the sample was placed in a sterile 96-well plate in a clean bench and the OD at 630 nm was measured using a microplate reader. At least three replicate wells were prepared for each group, and the experiment was repeated at least three times. The time-killing curve was drawn with the time point as the horizontal axis and the OD630 values ​​measured in different groups at different time points as the vertical axis.

[0030] The results of the time-kill curve experiment are shown in Figure 1The results showed that after 12 hours of combined use of methyl agrimony lactone and fluconazole, the combination group showed a good inhibitory effect on drug-resistant Candida albicans ATCC14053-FR compared with the control group.

[0031] Example 2 In vivo antifungal activity experiment of methyl agrimonolide 1. Experimental Methods After acclimating C57BL / 6 mice to the diet for 7 days, 96 mice were divided into Group 8 and housed in a 24 ± 2°C housing room with alternating day and night conditions for 12 hours. To simulate immunocompromised patients, all mice, except the blank and control groups, received cyclophosphamide (100 mg / kg) for three consecutive days. On day 4, mice in all groups, except the blank group, were injected with the resistant bacterium ATCC14053-FR via the tail vein to establish a fungal infection model. Two hours later, they were given drug treatments: caspofungin 5 mg / kg, 6-O-methyl agrimonolide (I) 52 mg / kg, fluconazole (FLC) 2 mg / kg, fluconazole and 6-O-methyl agrimonolide combined high (FLC + IH, 1:26) 54 mg / kg, and fluconazole and 6-O-methyl agrimonolide combined medium (FLC + IM, 1:13) 28 mg / kg. The drugs were then administered orally once daily for 8 consecutive days. Samples were collected on day 8 of treatment. Mice were anesthetized with 1% sodium pentobarbital, and blood was collected from the abdominal aorta. The thymus and spleen were removed and weighed. The right kidney and left upper lobe of the lung were fixed with paraformaldehyde for subsequent histological section preparation. At the conclusion of the experiment, data were collated, and immune organ markers and pathological sections were analyzed.

[0032] 2. Experimental Results See the results Figure 2 The results showed that compared with the blank group, the model group had the most significant weight loss, followed by the compound and fluconazole alone administration groups, while the combined drug group was able to maintain the weight of mice ( Figure 2 A). The thymus and spleen in the model group and fluconazole group were significantly reduced, while the combination group effectively alleviated the atrophy of the thymus and spleen at high concentrations ( Figure 2 B. Figure 2 C), indicating that the combined use of drugs can protect the immune system of mice against the invasion of drug-resistant Candida albicans. In addition, the pathological sections of the kidneys and lungs of mice showed that ( Figure 2 D, 2E), both the model group and the compound group showed extensive inflammatory cell infiltration and tissue hemorrhage, while the combination group reduced the lesion area, decreased inflammatory cell infiltration, and significantly improved renal tubular damage and alveolar edema.

[0033] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of a pharmaceutical composition in the preparation of a medicament for preventing and / or treating fungal infection, characterized in that: The pharmaceutical composition comprises 6-O-methyl agrimonolide or a pharmaceutically acceptable salt or solvent complex thereof and an azole antifungal drug.

2. The application according to claim 1, characterized in that The ratio of the 6-O-methyl agrimonolide or a pharmaceutically acceptable salt thereof, or a solvent complex to the azole antifungal drug is 1-50:1; Preferably, the ratio of the 6-O-methyl agrimonolide or a pharmaceutically acceptable salt thereof, or a solvent complex to the azole antifungal drug is 13-26:

1.

3. The application according to claim 1, characterized in that The azole antifungal drug is fluconazole.

4. The application according to claim 1, characterized in that The 6-O-methyl agrimony lactone is a racemate.

5. The application according to claim 1, characterized in that: The fungus is Candida albicans or drug-resistant Candida albicans.

6. The application according to claim 1, characterized in that: The drug can resist the invasion of drug-resistant Candida albicans by protecting the immune system function.

7. The use according to any one of claims 1 to 6, characterized in that: The form of the drug combination includes any one of the following forms: (1) preparing 6-O-methyl agrimonolide or a pharmaceutically acceptable salt or solvent complex thereof and an azole antifungal drug into independent preparations; (2) 6-O-methyl agrimony lactone or a pharmaceutically acceptable salt or solvent complex thereof and an azole antifungal drug are prepared into a compound preparation.

8. The use according to any one of claims 1 to 6, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that: The pharmaceutically acceptable excipients include any one or a combination of at least two of diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

10. The use according to any one of claims 1 to 6, characterized in that: The dosage form of the drug is tablet, capsule, granule, injection, oral liquid, pill, ointment, suspension, dispersion, syrup, suppository, gel, aerosol or patch.

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