An antifungal pharmaceutical composition based on (1,3)-beta-D-glucan synthase inhibitors

Abstract

The present application relates to the technical field of biological medicine, in particular to an antifungal drug composition based on (1,3)-beta-D-glucan synthase inhibitor and application thereof. In vivo and in vitro experiments prove that the combination of endogenous antibacterial peptide LL-37 and (1,3)-beta-D-glucan synthase inhibitor (such as caspofungin, micafungin, anidulafungin, rezafungin and albaconazole) shows significant synergistic inhibitory effect on multiple drug-resistant pathogenic fungi such as Candida albicans and Candida auris. Since free antibacterial peptides are easily degraded by proteases, the present application is based on the synergistic killing mechanism of breaking the network barrier of fungal cell wall and assisting endogenous defense peptide to target cell membrane, and ingeniously uses the "antibacterial peptide LL-37 expression promoter" to actively up-regulate the synthesis of host natural peptides, which provides a new drug regimen for clinical treatment of deep fungal infection.

Description

[0001] This application claims priority to an earlier patent application, CN 202511331862.7, filed on September 18, 2025, entitled "An antifungal pharmaceutical composition based on a (1,3)-β-D-glucan synthase inhibitor." The entire contents of the above-cited application are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedical technology, specifically to an antifungal drug composition based on a (1,3)-β-D-glucan synthase inhibitor. Background Technology

[0003] Fungal infections, especially invasive fungal infections, have long posed a serious threat to human health. Treatments such as organ transplantation, hematopoietic stem cell transplantation, cancer chemotherapy, and intravenous catheterization, or HIV infection, can all lead to weakened immunity, resulting in high morbidity and mortality rates from invasive fungal infections. Currently, standard drugs used clinically to treat invasive fungal infections mainly include amphotericin B, azoles, and echinocandins. Amphotericin B acts on fungal cell membrane lipids and binds to ergosterol, but its nephrotoxicity limits its use to large doses. Azole drugs, such as fluconazole and itraconazole, exert their antifungal effects by affecting the biosynthesis of ergosterol in the fungal cell membrane. They are inhibitors of lanosterol 14α-demethylase, but their main problems are high hepatotoxicity and nephrotoxicity, and severe drug resistance.

[0004] (1,3)-β-D-glucan synthase inhibitors are a new class of antifungal drugs. Currently marketed drugs mainly include echinocandins (such as caspofungin, micafungin, anidofungin, and lezafungin) and triterpenoids (such as eprifenacin). (1,3)-β-D-glucan synthase is a key enzyme in the synthesis of (1,3)-β-D-glucan in fungal cell walls. (1,3)-β-D-glucan synthase inhibitors disrupt fungal cell wall structure by blocking the synthesis of (1,3)-β-D-glucan. Since human cells do not have cell walls, these inhibitors have very low toxicity and can also be used to treat fungal infections resistant to azole drugs.

[0005] However, long-term use of (1,3)-β-D-glucan synthase inhibitors may lead to fungal resistance through target enzyme mutations or overexpression. At the same time, the high price also limits the application of (1,3)-β-D-glucan synthase inhibitors. Summary of the Invention

[0006] To overcome the technical challenges of increased fungal resistance and the inability of antimicrobial peptides to effectively penetrate cell walls in existing technologies, this invention provides a novel "host-pathogen bidirectional regulation" antifungal strategy.

[0007] This invention is based on a groundbreaking discovery: the direct addition of the antimicrobial peptide LL-37, or the specific upregulation of the expression level of the endogenous antimicrobial peptide LL-37 in host cells by an antimicrobial peptide LL-37 expression promoter, while (1,3)-β-D-glucan synthase inhibitors can disrupt the integrity of fungal cell walls. When these two are used in combination, the damaged cell wall can no longer block the entry of LL-37, allowing high concentrations of the antimicrobial peptide LL-37 to penetrate and destroy the fungal cell membrane, thereby producing a significant synergistic lethal effect.

[0008] Based on this mechanism, the present invention provides an antifungal drug composition based on (1,3)-β-D-glucan synthase inhibitor.

[0009] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the invention provides the use of antimicrobial peptide LL-37 or an antimicrobial peptide LL-37 expression promoter in combination with a (1,3)-β-D-glucan synthase inhibitor in the preparation of antifungal drugs; The mass ratio of the antimicrobial peptide LL-37 to the (1,3)-β-D-glucan synthase inhibitor is (1~5000):1; The mass ratio of the antimicrobial peptide LL-37 expression promoter to the (1,3)-β-D-glucan synthase inhibitor is 1:(1~5×10⁻⁶). 6 ).

[0010] In one or more embodiments, the mass ratio of the antimicrobial peptide LL-37 to the (1,3)-β-D-glucan synthase inhibitor is (1~4000):1, preferably (5~3200):1. Further, when the antimicrobial peptide LL-37 is used in combination with the (1,3)-β-D-glucan synthase inhibitor, the FICI ≤ 0.5.

[0011] The mass ratio of the antimicrobial peptide LL-37 expression promoter to the (1,3)-β-D-glucan synthase inhibitor is 1:(10~4.6×10). 6 ), preferably 1: (100~4×10 6 ), further preferably 1: (1000~3×10 6 Furthermore, when the antimicrobial peptide LL-37 expression promoter is used in combination with a (1,3)-β-D-glucan synthase inhibitor, the FICI ≤ 0.5.

[0012] In one or more embodiments, the use of antimicrobial peptide LL-37 in combination with (1,3)-β-D-glucan synthase inhibitor in the preparation of antifungal drugs.

[0013] In one or more embodiments, the use of an antimicrobial peptide LL-37 expression promoter in combination with a (1,3)-β-D-glucan synthase inhibitor in the preparation of an antifungal drug.

[0014] In one or more embodiments, the amino acid sequence of the antimicrobial peptide LL-37 is shown in SEQ ID NO: 1 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES).

[0015] In one or more embodiments, the expression promoter of the antimicrobial peptide LL-37 is vitamin D or a derivative thereof; Preferably, the vitamin D or its derivatives include one or more of the following: vitamin D2, vitamin D3, doxercalciferol, calcidiol, calcidiol monohydrate, alfacalcidol, inecalcitol, calcipotriol, calcipotriol monohydrate, calcitriol, maxacalcitol, ercalcitriol, paricalcitol, eldecalcitol, tacalcitol, and falecalcitriol, preferably calcitriol.

[0016] In one or more embodiments, (1,3)-β-D-glucan synthase inhibitors include one or more of echinocandins and triterpenoids; Preferably, the echinocandins include one or more of caspofungin, micafungin, anidulafungin, and rezafungin; Preferably, the triterpenoid compound includes Ibrexafungerp.

[0017] In one or more embodiments, the fungus includes one or more of Candida albicans, Candida auris, Candida tropicalis, Candida glabrata, Candida parapsilosis, and Candida krusei.

[0018] A second aspect of the present invention provides an antifungal drug comprising antimicrobial peptide LL-37 or an antimicrobial peptide LL-37 expression promoter and (1,3)-β-D-glucan synthase inhibitor.

[0019] In one or more embodiments, the antifungal drug includes the antimicrobial peptide LL-37 and a (1,3)-β-D-glucan synthase inhibitor; The mass ratio of the antimicrobial peptide LL-37 to the (1,3)-β-D-glucan synthase inhibitor is (1~5000):1. Furthermore, when the antimicrobial peptide LL-37 is used in combination with the (1,3)-β-D-glucan synthase inhibitor, the FICI ≤ 0.5. In one or more embodiments, the antifungal drug includes an antimicrobial peptide LL-37 expression promoter and a (1,3)-β-D-glucan synthase inhibitor; The mass ratio of the antimicrobial peptide LL-37 expression promoter to the (1,3)-β-D-glucan synthase inhibitor is 1:(1~5×10⁻⁶). 6 The preferred ratio is 1: (10~4.6×10). 6 ), further preferably 1: (100~4×10 6 ), and even more preferably 1: (1000~3×10 6 Furthermore, when the antimicrobial peptide LL-37 expression promoter is used in combination with a (1,3)-β-D-glucan synthase inhibitor, the FICI ≤ 0.5.

[0020] In one or more embodiments, the amino acid sequence of the antimicrobial peptide LL-37 is shown in SEQ ID NO: 1.

[0021] In one or more embodiments, the expression promoter of the antimicrobial peptide LL-37 is vitamin D or a derivative thereof; Preferably, the vitamin D or its derivatives include one or more of the following: vitamin D2, vitamin D3, doxercalciferol, calcidiol, calcidiol monohydrate, alfacalcidol, inecalcitol, calcipotriol, calcipotriol monohydrate, calcitriol, maxacalcitol, ercalcitriol, paricalcitol, eldecalcitol, tacalcitol, and falecalcitriol, preferably calcitriol.

[0022] In one or more embodiments, (1,3)-β-D-glucan synthase inhibitors include one or more of echinocandins and triterpenoids; Preferably, the echinocandin class includes one or more of caspofungin, micafungin, anidulafungin, and rezafungin; Preferably, the triterpenoid compound includes Ibrexafungerp.

[0023] In one or more embodiments, the fungus includes one or more of Candida albicans, Candida auris, Candida tropicalis, Candida glabrata, Candida parapsilosis, and Candida krusei.

[0024] A third aspect of the invention provides a pharmaceutical composition comprising the antifungal drug described in the second aspect.

[0025] In one or more embodiments, the pharmaceutical composition further includes pharmaceutically acceptable carriers, excipients, and diluents.

[0026] The non-pharmaceutical active ingredients that may be included, such as carriers, excipients, and diluents, are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0027] Preferably, the carrier, excipients, and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0028] Preferably, the dosage form of the drug is a suspension, emulsion, granules, spray, injection, transdermal absorbent, a dosage form suitable for transfection, tablet, powder, granules or capsule.

[0029] The medicament of the present invention can be administered into the body by known means, such as intravenous systemic delivery or local injection to the tissue of interest. Alternatively, it can be administered via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via a single dose or multiple doses. Those skilled in the art will understand that the actual dose to be administered in the present invention can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue thereof, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0030] Preferably, the drug can be administered to humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, and chimpanzees.

[0031] A fourth aspect of the present invention provides an antifungal kit comprising: (1) A first container containing a (1,3)-β-D-glucan synthase inhibitor or a pharmaceutically acceptable carrier, excipient, and diluent thereof; and (2) A second container containing an antimicrobial peptide LL-37 expression promoter or its pharmaceutically acceptable carrier, excipient and diluent; Preferably, the kit further includes instructions for use, which instruct the combined administration of the (1,3)-β-D-glucan synthase inhibitor and the antimicrobial peptide LL-37 expression promoter, wherein the mass ratio of the antimicrobial peptide LL-37 expression promoter to the (1,3)-β-D-glucan synthase inhibitor is preferably 1:(1~5×10⁻⁶). 6 ).

[0032] The beneficial effects of this invention are as follows: (1) In vitro experimental results in this invention show that the antimicrobial peptide LL-37, when used in combination with (1,3)-β-D-glucan synthase inhibitors (caspofungin, micafungin, anidofungin, lezafungin, and eprifenacin), has a synergistic inhibitory effect on Candida albicans, Candida auris, Candida tropicalis, Candida glabrata, Candida parapsilosis, and Candida krusei. Therefore, the antimicrobial peptide LL-37 can be combined with (1,3)-β-D-glucan synthase inhibitors to prepare antifungal drugs. However, the antimicrobial peptide LL-37 is easily degraded by proteases in vivo. Therefore, this invention uses an antimicrobial peptide LL-37 expression promoter to upregulate the expression of the antimicrobial peptide LL-37 in vivo, and synergistically inhibits the antibacterial activity with (1,3)-β-D-glucan synthase inhibitors in vivo.

[0033] (2) The present invention uses antimicrobial peptide LL-37 or antimicrobial peptide LL-37 expression promoter in combination with (1,3)-β-D-glucan synthase inhibitor, which can effectively reduce the minimum effective inhibitory concentration of (1,3)-β-D-glucan synthase inhibitor and reduce the dosage of (1,3)-β-D-glucan synthase inhibitor, thereby overcoming the problem of fungal resistance in clinical practice.

[0034] (3) Developing antifungal treatment regimens based on (1,3)-β-D-glucan synthase inhibitors not only enriches the existing treatment methods of antifungal drugs, but also provides a new, effective and safe strategy for the clinical treatment of complex fungal infections, which has important scientific significance and broad application prospects.

[0035] (4) Through multi-dimensional physicochemical characterization experiments, this invention verified the "double-hit" synergistic effect of LL-37 and (1,3)-β-D-glucan synthase inhibitor at the molecular mechanism level: Zeta potential measurement confirmed that the surface charge of fungal cells changed from negative to positive after the combined treatment, indicating that the positively charged LL-37 effectively adsorbed onto the cell membrane surface exposed due to cell wall destruction; DiSC3 (5) fluorescent probe detection confirmed that the combined treatment led to significant cell membrane depolarization, indicating that LL-37 disrupted the electrochemical properties of the cell membrane. The gradient of the enzyme was studied; transmission electron microscopy directly observed that the use of (1,3)-β-D-glucan synthase inhibitor alone caused the cell wall boundary to become blurred, while the combination of the two resulted in complete cell wall disintegration and cytoplasmic leakage; fluorescently labeled LL-37 verified that caspofungin could promote the focusing of LL-37 on the fungal cell membrane; caspofungin caused the outward turning of negatively charged phospholipids on the surface of fungal cells, and this outward turning of negatively charged phospholipids showed a dose-response relationship with caspofungin, and the negatively charged phospholipids further promoted the above-observed focusing of LL-37 on the fungal cell membrane.

[0036] The above mechanism verification experiments provide a solid scientific basis for the synergistic effect of the antimicrobial peptide LL-37 or its expression promoter in combination with the (1,3)-β-D-glucan synthase inhibitor in the claims of this invention. Attached Figure Description

[0037] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0038] Figure 1 The images show the antifungal effects of LL-37 combined with caspofungin. In the images, a represents the effect of LL-37 combined with caspofungin against Candida albicans SC5314, and b represents the effect of LL-37 combined with caspofungin against Candida auris CBS12774. Figure 2 The images show the antifungal effects of LL-37 combined with caspofungin, where a represents the effect of LL-37 combined with caspofungin against Candida tropicalis CT2; and b represents the effect of LL-37 combined with caspofungin against Candida glabrata CG1. Figure 3 The images show the antifungal effects of LL-37 combined with caspofungin, where a represents the effect of LL-37 combined with caspofungin against Candida parapsilosis CP1; and b represents the effect of LL-37 combined with caspofungin against Candida krusei CK1. Figure 4 The images show the antifungal effects of LL-37 combined with micafungin. In the images, a represents the effect of LL-37 combined with micafungin against Candida albicans SC5314, and b represents the effect of LL-37 combined with micafungin against Candida auris CBS12774. Figure 5 The images show the antifungal effects of LL-37 combined with micafungin, where a represents the effect of LL-37 combined with micafungin against Candida tropicalis CT2; and b represents the effect of LL-37 combined with micafungin against Candida glabrata CG1. Figure 6 The images show the antifungal effects of LL-37 combined with micafungin, where a represents the effect of LL-37 combined with micafungin against Candida parapsilosis CP1; and b represents the effect of LL-37 combined with micafungin against Candida krusei CK1. Figure 7 The images show the antifungal effects of LL-37 combined with anidoxane, where a represents the effect of LL-37 combined with anidoxane against Candida albicans SC5314, and b represents the effect of LL-37 combined with anidoxane against Candida auris CBS12774. Figure 8 The images show the antifungal effects of LL-37 combined with anidoxane, where a represents the effect of LL-37 combined with anidoxane against Candida tropicalis CT2; and b represents the effect of LL-37 combined with anidoxane against Candida glabrata CG1. Figure 9 The images show the antifungal effects of LL-37 combined with anidoxurine, where a represents the effect of LL-37 combined with anidoxurine against Candida parapsilosis CP1; and b represents the effect of LL-37 combined with anidoxurine against Candida krusei CK1. Figure 10 The images show the antifungal effects of LL-37 combined with Erifenjin, where a represents the effect of LL-37 combined with Erifenjin against Candida albicans SC5314, and b represents the effect of LL-37 combined with Erifenjin against Candida auris CBS12774. Figure 11 The images show the antifungal effects of LL-37 combined with irifenol, where a represents the antifungal effect of LL-37 combined with irifenol against Candida tropicalis CT2; and b represents the antifungal effect of LL-37 combined with irifenol against Candida glabrata CG1. Figure 12 The images show the antifungal effects of LL-37 combined with irifentanil, where a represents the effect of LL-37 combined with irifentanil against Candida parapsilosis CP1; and b represents the effect of LL-37 combined with irifentanil against Candida krusei CK1. Figure 13 The study aimed to assess the synergistic effect of low-dose caspofungin and calcitriol against fungal infection in mice. In the figures, a represents the bacterial load in mouse kidneys after treatment with low-dose caspofungin; b represents the glycogen (PAS) staining results in mouse kidney sections; and c represents the hematoxylin-eosin (HE) staining results in mouse kidney sections. Figure 14 The study aimed to evaluate the synergistic effect of therapeutic doses of caspofungin and calcitriol against fungal infection in mice. In the figures, a represents the bacterial load in mouse kidneys after treatment with therapeutic doses of caspofungin; b represents the results of PAS staining of mouse kidney sections; and c represents the results of HE staining of mouse kidney sections.

[0039] Figure 15 To verify the synergistic antifungal mechanism of LL-37 combined with caspofungin, a) shows the Zeta potential measurement results of Candida albicans SC5314 under different treatment conditions, with the ordinate representing the Zeta potential value (mV); b) shows the changes in cell membrane potential of Candida albicans SC5314 under different treatment conditions, detected by the DiSC3(5) fluorescent probe, with the ordinate representing the relative fluorescence intensity; c) shows the transmission electron microscope (TEM) images of Candida albicans SC5314 under different treatment conditions, respectively showing the ultrastructural changes of cells in the blank control group, the caspofungin-only (0.01 μg / mL) group, the LL-37-only (1 μg / mL) group, and the combined group.

[0040] Figure 16Caspofungin exerts a synergistic antifungal effect by promoting the focusing of LL-37 on the cell membrane through increasing the extravasation of negatively charged phospholipids. Specifically, a) caspofungin promotes the focusing of fluorescently labeled LL-37 on the cell membrane; b) caspofungin dose-dependently increases the extravasation of negatively charged phospholipids. Detailed Implementation

[0041] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0042] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0043] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0044] Antimicrobial peptide LL-37 was purchased from Anhui Guoping Pharmaceutical Co., Ltd. Caspofungin (CAP), micafungin, anidulafungin, rezafungin, and ibrexafungerp citrate were purchased from Yuanye Biotechnology. Vitamin D2, Vitamin D3, Doxercalciferol, Calcifediol, Calcifediol Monohydrate, Alfacalcidol, Inecalcitol, Calcipotriol, Calcipotriol Monohydrate, Calcitriol, Maxacalcitol, Ercalcitriol, Paricalcitol, Eldecalcitol, Tacalcitol, and Falecalcitriol were all purchased from MCE. Cell RNA extraction kit (Beyozol) was purchased from Beyotime. The SPARKscript® II All-in-one RT SuperMix for qPCR Reverse Transcription Kit was purchased from Cisco. Pfeiffer: Human diffuse large B-cell lymphoma.

[0045] Example 1 The fungal cells cultured overnight were diluted to 1×10⁻⁶ using RPMI 1640 medium. 3 CFU / mL, working solutions of caspofungin, micafungin, anidofungin, lezafungin, and eprifenofungin were prepared using diluted bacterial suspensions at different drug concentration gradients. Different concentrations of working solution were added to each column of a 96-well plate, and a certain concentration of antimicrobial peptide LL-37 working solution was added to each well in rows A-G and serially diluted. After static incubation at 30 ℃ for 24 h, the OD value of each well was measured at 600 nm. Control wells were blank bacterial suspensions without added drugs. The growth rate of each well was calculated as (drug-added well value / control well value) × 100%, with the growth rate of control wells being 100%. The drug concentration in wells where the absorbance decreased by 80% compared to the control wells was defined as the minimum inhibitory concentration (MIC).

[0046] Synergistic effect evaluation: The combined effects of antimicrobial drugs in vitro or in vivo can exhibit four types of effects: "unrelated," "additive," "synergistic," and "antagonistic." The results of drug susceptibility testing for combined use are assessed using the fractional inhibitory concentration index (FIC) to determine the interaction effect of the two drugs.

[0047] Fractional inhibitory concentration index (FICI) method, the formula is as follows: ΣFICI=FIC A +FICB =C A / MIC A +C B / MIC B ; Wherein, FICI is the fractional inhibitory concentration index, FIC A and FIC B These represent the MIC value of drugs A and B when used in combination, divided by the MIC values ​​of drugs A and B when used alone. A and MIC B These are the minimum inhibitory concentrations (MICs) of drugs A and B when used alone, and C. A With C B FICI represents the concentrations of each drug required to achieve the same therapeutic effect when used in combination. FICI > 4 indicates antagonistic effect, FICI between 0.5 and 4 indicates additive or indifferent effect, and FICI ≤ 0.5 is defined as synergistic effect.

[0048] The antifungal effect of antimicrobial peptide LL-37 in combination with caspofungin, micafungin, anidoxane, lezafenamicin, and eprifenamicin is as follows: Figures 1-12 And as shown in Tables 1 and 2.

[0049] Table 1 Results of combined medication

[0050] Table 2 Results of combined medication

[0051] As shown in Tables 1 and 2, the antimicrobial peptide LL-37 alone had no inhibitory effect on Candida albicans SC5314, Candida auris CBS12774, Candida tropicalis CT2, Candida glabrata CG1, Candida parapsilosis CP1, and Candida krusei CK1, with a minimum inhibitory concentration greater than 128 μg / mL.

[0052] The minimum inhibitory concentrations (MICs) of caspofungin alone against Candida albicans SC5314, Candida auris CBS12774, Candida tropicalis CT2, Candida glabrata CG1, Candida parapsilosis CP1, and Candida krusei CK1 were 0.25 μg / mL, 1 μg / mL, 1 μg / mL, 1 μg / mL, 2 μg / mL, and 2 μg / mL, respectively. When antimicrobial peptide LL-37 and caspofungin were used in combination, the FICI values ​​were all less than 0.5, indicating that caspofungin and antimicrobial peptide LL-37 had a significant synergistic effect. When used in combination with antimicrobial peptide LL-37, caspofungin achieved a minimum inhibitory concentration (MIC) of 0.015626 μg / mL against Candida albicans SC5314, 0.015626 μg / mL against Candida auris CBS12774, 0.001953 μg / mL against Candida tropicalis CT2, 0.003906 μg / mL against Candida glabrata CG1, 0.03125 μg / mL against Candida parapneumoniae CP1, and 0.03125 μg / mL against Candida krusei CK1.

[0053] The minimum inhibitory concentrations (MICs) of micafungin alone against Candida albicans SC5314, Candida auris CBS12774, Candida tropicalis CT2, Candida glabrata CG1, Candida parapsilosis CP1, and Candida krusei CK1 were 0.03125 μg / mL, 0.5 μg / mL, 0.0625 μg / mL, 0.03125 μg / mL, 1 μg / mL, and 0.125 μg / mL, respectively. When antimicrobial peptide LL-37 and micafungin were used in combination, the FICI values ​​were all less than 0.5, indicating that micafungin and antimicrobial peptide LL-37 have a significant synergistic effect. When used in combination with antimicrobial peptide LL-37, the minimum inhibitory concentration (MIC) of micafungin against Candida albicans SC5314 reached 0.001953 μg / mL, against Candida auris CBS12774 reached 0.03125 μg / mL, against Candida tropicalis CT2 reached 0.003906 μg / mL, against Candida glabrata CG1 reached 0.001953 μg / mL, against Candida parapsilosis CP1 reached 0.0625 μg / mL, and against Candida krusei CK1 reached 0.007813 μg / mL.

[0054] The minimum inhibitory concentrations (MICs) of anidoxurine alone against Candida albicans SC5314, Candida auris CBS12774, Candida tropicalis CT2, Candida glabrata CG1, Candida parapsilosis CP1, and Candida krusei CK1 were 0.007813 μg / mL, 0.125 μg / mL, 0.015625 μg / mL, 0.03125 μg / mL, 1 μg / mL, and 0.5 μg / mL, respectively. When antimicrobial peptide LL-37 and anifenin were used in combination, the FICI values ​​were all less than 0.5, indicating that anifenin and antimicrobial peptide LL-37 had a significant synergistic effect. When used in combination with antimicrobial peptide LL-37, anifenin achieved a minimum inhibitory concentration (MIC) of 0.001953 μg / mL against Candida albicans SC5314, 0.001953 μg / mL against Candida auris CBS12774, 0.001953 μg / mL against Candida tropicalis CT2, 0.001953 μg / mL against Candida glabrata CG1, 0.0625 μg / mL against Candida parapsilosis CP1, and 0.125 μg / mL against Candida krusei CK1.

[0055] The minimum inhibitory concentrations (MICs) of eryrifencanil alone against Candida albicans SC5314, Candida auris CBS12774, Candida tropicalis CT2, Candida glabrata CG1, Candida parapsilosis CP1, and Candida krusei CK1 were 0.125 μg / mL, 1 μg / mL, 1 μg / mL, 2 μg / mL, 1 μg / mL, and 2 μg / mL, respectively. When antimicrobial peptide LL-37 and irifenol were used in combination, the FICI values ​​were all less than 0.5, indicating that irifenol and antimicrobial peptide LL-37 had a significant synergistic effect. When used in combination with antimicrobial peptide LL-37, the minimum inhibitory concentration (MIC) of irifenol against Candida albicans SC5314 reached 0.0078125 μg / mL, against Candida auris CBS12774 reached 0.0625 μg / mL, against Candida tropicalis CT2 reached 0.125 μg / mL, against Candida glabrata CG1 reached 0.25 μg / mL, against Candida parapsilosis CP1 reached 0.125 μg / mL, and against Candida krusei CK1 reached 0.25 μg / mL.

[0056] The minimum inhibitory concentrations (MICs) of lezafenib alone against Candida albicans SC5314, Candida auris CBS12774, Candida tropicalis CT2, Candida glabrata CG1, Candida parapsilosis CP1, and Candida krusei CK1 were 0.015625 μg / mL, 0.25 μg / mL, 0.03125 μg / mL, 0.0625 μg / mL, 0.5 μg / mL, and 0.25 μg / mL, respectively. When antimicrobial peptide LL-37 and lezafenamic acid were used in combination, the FICI values ​​were all less than 0.5, indicating that lezafenamic acid and antimicrobial peptide LL-37 had a significant synergistic effect. When used in combination with antimicrobial peptide LL-37, the minimum inhibitory concentration (MIC) of lezafenamic acid against Candida albicans SC5314 reached 0.001953 μg / mL, against Candida auris CBS12774 reached 0.015625 μg / mL, against Candida tropicalis CT2 reached 0.003906 μg / mL, against Candida glabrata CG1 reached 0.003906 μg / mL, against Candida parapsilosis CP1 reached 0.03125 μg / mL, and against Candida krusei CK1 reached 0.03125 μg / mL.

[0057] In summary, the combination of antimicrobial peptide LL-37 with (1,3)-β-D-glucan synthase inhibitors (caspofungin, micafungin, anidoxane, lezafungin, and eprifenacin) can effectively reduce the minimum effective concentration (MID) of (1,3)-β-D-glucan synthase inhibitors and the dosage of (1,3)-β-D-glucan synthase inhibitors, thereby overcoming the problem of fungal resistance in clinical practice.

[0058] Example 2 Antimicrobial peptide LL-37 is an active peptide generated by enzymatic cleavage of the precursor protein hCAP-18 (human cationic antimicrobial protein 18) encoded by the cyclic adenosine monophosphate (cAMP) gene. It is the only human member of the cathelicidin family. LL-37 is expressed in various cell types, particularly neutrophils, keratinocytes, and epithelial cells, primarily stored intracellularly as a granule protein. Upon activation, the cathelicin domain is cleaved by serine protease 3, releasing the active peptide. However, LL-37 is readily degraded by proteases in vivo. Therefore, this invention employs an antimicrobial peptide LL-37 expression promoter to upregulate its expression in vivo.

[0059] Screening for promoters of antimicrobial peptide LL-37 expression: The experimental procedure is as follows: (1) Sample preparation: Adjust Pfeiffer cells in logarithmic growth phase to a density of 2×10⁻⁶. 5Cells / mL; seeded into 24-well plates, added 100 nM of the reagent to each well, and set up a blank control group; placed the 24-well plates in an incubator at 37 ℃ with 5% CO2 for 24 h; (2) RNA extraction: After incubation, RNA was extracted from cells according to the Beyozol kit method; (3) Reverse transcription: RNA was reverse transcribed into cDNA using the SPARKscript® II All-in-one RT SuperMix for qPCR Reverse Transcription Kit; (4) Real-time quantitative PCR: β-actin was used as an internal standard gene, and the expression of the cAMP gene was measured using the Eppendorf Mastercycler real-time quantitative PCR system; the specific primers are as follows: h-βactin-F (SEQ ID NO: 2):AGTTGCGTTACACCCTTTC; h-βactin-R (SEQ ID NO: 3): CCTTCACCGTTCCAGTTT; hCAMP-F (SEQ ID NO: 4):GACACAGCAGTCACCAGAGGAT; hCAMP-R (SEQ ID NO: 5):TCACAACTGATGTCAAAGGAGCC; The experimental results are shown in Table 3. As can be seen from Table 3, vitamin D or its derivatives can increase the expression level of CAMP gene in Pfeiffer cell lines to varying degrees. Among them, the expression level of CAMP gene in Pfeiffer cell lines induced by calcitriol is the highest.

[0060] Table 3. Effects of Vitamin D or its derivatives on cAMP gene expression.

[0061] Example 3 Low-dose caspofungin and calcitriol synergistically combat fungal infections in mice: Eight-week-old male Balb / c mice were randomly divided into four groups of three mice each: a low-dose caspofungin monotherapy group, a calcitriol monotherapy group, a combination therapy group, and a control group. Overnight Candida albicans SC5314 cells were diluted to 5 × 10⁻⁶ mcg using physiological saline. 6CFU / mL; 100 μL of cell suspension was injected into each mouse via the tail vein. 24 h after infection, different drugs were injected intraperitoneally. Based on body weight, the caspofungin monotherapy group received 0.1 mg / kg caspofungin, the calcitriol monotherapy group received 1 μg / kg calcitriol, the combination group received a mixture of 0.1 mg / kg caspofungin and 1 μg / kg calcitriol, and the control group received the same volume of physiological saline. Administration was repeated once daily for 3 consecutive days. On day 4, the mice were euthanized, and their kidneys were dissected. One kidney was fixed in 4% paraformaldehyde for PAS and HE staining analysis; the other kidney was ground and plate-treated to calculate the bacterial load.

[0062] Experimental results are as follows Figure 13 As shown, from Figure 13 As can be seen, calcitriol can significantly enhance the antifungal activity of caspofungin against Candida albicans SC5314 in vivo. In this example, the low dose is the concentration at which caspofungin cannot produce a therapeutic effect. However, by using it in combination with calcitriol, the renal bacterial load was significantly reduced compared with the low-dose caspofungin monotherapy group, the control group, and the calcitriol monotherapy group. This indicates that when caspofungin does not produce a therapeutic effect in vivo, the combination with calcitriol can produce a therapeutic effect.

[0063] Example 4 Therapeutic doses of caspofungin and calcitriol synergistically combat fungal infections in mice: Eight-week-old male Balb / c mice were randomly divided into four groups of three mice each: caspofungin monotherapy group, calcitriol monotherapy group, combination therapy group, and control group. Overnight Candida albicans SC5314 cells were diluted to 5 × 10⁻⁶ mcg using physiological saline. 6 CFU / mL; 100 μL of cell suspension was injected into the tail vein of each mouse. 24 h after infection, different drugs were injected intraperitoneally. Mice were administered drugs according to body weight. The caspofungin monotherapy group received caspofungin at a concentration of 0.3 mg / kg, the calcitriol monotherapy group received calcitriol at a concentration of 1 μg / kg, the combination therapy group received a mixture of 0.3 mg / kg caspofungin and 1 μg / kg calcitriol, and the control group received the same volume of physiological saline. Drug administration was repeated once daily for 3 consecutive days. On the 4th day, the mice were euthanized, and the kidneys were dissected and harvested. One kidney was fixed in 4% paraformaldehyde for PAS and HE staining analysis; the other kidney was ground and plate-treated to calculate the bacterial load.

[0064] Experimental results are as follows Figure 14 As shown, from Figure 14As can be seen, calcitriol can significantly enhance the antifungal activity of caspofungin against Candida albicans SC5314 in vivo. The therapeutic dose of caspofungin is the concentration commonly used in laboratory caspofungin treatment. After being used in combination with calcitriol, the renal bacterial load was significantly reduced compared with the caspofungin monotherapy group, the calcitriol monotherapy group, and the blank group. This indicates that while caspofungin produces a normal therapeutic effect, the effect is enhanced when used in combination with calcitriol, achieving a synergistic effect.

[0065] Example 5 Therapeutic doses of caspofungin and calcitriol synergistically combat fungal infections in mice: Compared with Example 4, the fungi in mice were replaced with Candida auris CBS12774, Candida tropicalis CT2, Candida glabrata CG1, Candida parapsilosis CP1, and Candida krusei CK1. The specific dosages of caspofungin monotherapy, calcitriol, combination therapy, and control group were the same as in Example 4, and the other experimental procedures were exactly the same as in Example 4. The antibacterial results are shown in Table 4.

[0066] Table 4. Synergistic antifungal effects of caspofungin and calcitriol

[0067] Example 6 Therapeutic doses of caspofungin and vitamin D or its derivatives synergistically combat infection in mice: Compared with Example 4, the fungus in mice was still Candida albicans SC5314. Calcitriol was replaced with other vitamin D or its derivatives. The concentration of caspofungin in the caspofungin monotherapy group was 0.3 mg / kg, the concentration of vitamin D or its derivatives in the vitamin D or its derivative monotherapy group was 1 μg / kg, the combination group was injected with a mixed solution of 0.3 mg / kg caspofungin and 1 μg / kg vitamin D or its derivatives, and the control group was injected with the same volume of physiological saline. Other experimental procedures were exactly the same as in Example 4. The antibacterial results are shown in Table 5.

[0068] Table 5. Synergistic antifungal effects of caspofungin and vitamin D or its derivatives.

[0069] Example 7 Therapeutic doses of micafungin and vitamin D or its derivatives synergistically combat infection in mice: Compared with Example 4, the fungus in mice was still Candida albicans SC5314. Caspofungin was replaced with micafungin. The concentration of micafungin injected in the micafungin monotherapy group was 0.3 mg / kg, the concentration of vitamin D or its derivative injected in the vitamin D or its derivative monotherapy group was 1 μg / kg, the combination group was injected with a mixed solution of 0.3 mg / kg micafungin and 1 μg / kg vitamin D or its derivative, and the control group was injected with the same volume of physiological saline. Other experimental procedures were exactly the same as in Example 4. The antibacterial results are shown in Table 6.

[0070] Table 6. Synergistic antifungal effects of micafungin and vitamin D or its derivatives.

[0071] Example 8 Therapeutic doses of anidoxane and vitamin D or its derivatives synergistically combat infection in mice: Compared with Example 4, the fungus in mice was still Candida albicans SC5314. Caspofungin was replaced with anidoxurine. The anidoxurine monotherapy group was injected with anidoxurine concentration of 0.3 mg / kg, the vitamin D or its derivative monotherapy group was injected with vitamin D or its derivative at a concentration of 1 μg / kg, the combination group was injected with a mixed solution of 0.3 mg / kg anidoxurine and 1 μg / kg vitamin D or its derivative, and the control group was injected with the same volume of physiological saline. Other experimental procedures were exactly the same as in Example 4. The antibacterial results are shown in Table 7.

[0072] Table 7 Synergistic antifungal effects of anifenidine and vitamin D or its derivatives

[0073] Example 9 Therapeutic doses of anidoxane and calcitriol synergistically combat fungal infections in mice: Compared with Example 8, the fungi in mice were replaced with Candida auris CBS12774, Candida tropicalis CT2, Candida glabrata CG1, Candida parapsilosis CP1, and Candida krusei CK1. The concentration of anidoxurine in the anidoxurine monotherapy group was 0.3 mg / kg, the concentration of calcitriol in the calcitriol monotherapy group was 1 μg / kg, the combination therapy group was injected with a mixed solution of 0.3 mg / kg anidoxurine and 1 μg / kg calcitriol, and the control group was injected with the same volume of physiological saline. Other experimental procedures were exactly the same as in Example 4. The antibacterial results are shown in Table 8.

[0074] Table 8 Synergistic antifungal effects of anidoxurine and calcitriol

[0075] Example 10 Therapeutic doses of irifentanyl and vitamin D or its derivatives synergistically combat infection in mice: Compared with Example 4, the fungus in mice was still Candida albicans SC5314. Caspofungin was replaced with irifenol. In the irifenol monotherapy group, the injection concentration of irifenol was 0.3 mg / kg. In the vitamin D or its derivative monotherapy group, the injection concentration of vitamin D or its derivative was 1 μg / kg. The combination group was injected with a mixed solution of 0.3 mg / kg irifenol and 1 μg / kg vitamin D or its derivative. The control group was injected with the same volume of physiological saline. Other experimental procedures were exactly the same as in Example 4. The antibacterial results are shown in Table 9.

[0076] Table 9. Synergistic antifungal effects of irifenol and vitamin D or its derivatives.

[0077] Example 11 Therapeutic doses of erythromycin and calcitriol synergistically combat fungal infections in mice: Compared with Example 10, the fungi in mice were replaced with Candida auris CBS12774, Candida tropicalis CT2, Candida glabrata CG1, Candida parapsilosis CP1, and Candida krusei CK1. The concentration of erifenin in the erifenin monotherapy group was 0.3 mg / kg, the concentration of calcitriol in the calcitriol monotherapy group was 1 μg / kg, the combination group was injected with a mixed solution of 0.3 mg / kg erifenin and 1 μg / kg calcitriol, and the control group was injected with the same volume of physiological saline. Other experimental procedures were exactly the same as in Example 4. The antibacterial results are shown in Table 10.

[0078] Table 10 Synergistic antifungal effects of irifenazole and calcitriol

[0079] Example 12 Therapeutic doses of lizafenib and vitamin D or its derivatives synergistically combat infection in mice: Compared with Example 4, the fungus in mice was still Candida albicans SC5314. Caspofungin was replaced with lezafungin. In the lezafungin monotherapy group, the lezafungin injection concentration was 0.3 mg / kg. In the vitamin D or its derivative monotherapy group, the vitamin D or its derivative injection concentration was 1 μg / kg. The combination group was injected with a mixed solution of 0.3 mg / kg lezafungin and 1 μg / kg vitamin D or its derivative. The control group was injected with the same volume of physiological saline. Other experimental procedures were exactly the same as in Example 4. The antibacterial results are shown in Table 11.

[0080] Table 11 Synergistic antifungal effects of lezafenib and vitamin D or its derivatives

[0081] Example 13 Therapeutic doses of lezafenib and calcitriol synergistically combat fungal infections in mice: Compared with Example 12, the fungi in mice were replaced with Candida auris CBS12774, Candida tropicalis CT2, Candida glabrata CG1, Candida parapsilosis CP1, and Candida krusei CK1. The concentration of lezafenamic acid injected in the lezafenamic acid monotherapy group was 0.3 mg / kg, the concentration of calcitriol injected in the calcitriol monotherapy group was 1 μg / kg, the combination group was injected with a mixed solution of 0.3 mg / kg lezafenamic acid and 1 μg / kg calcitriol, and the control group was injected with the same volume of physiological saline. Other experimental procedures were exactly the same as in Example 4. The antibacterial results are shown in Table 12.

[0082] Table 12 Synergistic antifungal effects of lezafenib and calcitriol

[0083] Based on the results of Examples 4 to 13, it can be seen that (1,3)-β-D-glucan synthase inhibitors, including caspofungin, micafungin, anidofungin, irifenfungin, and lezafenfungin, can achieve synergistic antifungal effects when used in combination with vitamin D or its derivatives, including vitamin D2, vitamin D3, calciferol, calcidiol, calcidiol monohydrate, alfacalciferol, calcipotriol, calcipotriol monohydrate, calciferol, inelcalciferol, mascalcalciferol, ircalcitriol, paricalciferol, edicalciferol, tacalciferol, and falicalciferol. Among these, calciferol is the most preferred in treatment at the same dose. Caspofungin, micafungin, anidofungin, eprazole, and lezafungin can all be used in combination with calcitriol to reduce the bacterial load of Candida albicans SC5314, Candida auris CBS12774, Candida tropicalis CT2, Candida glabrata CG1, Candida parapsilosis CP1, and Candida krusei CK1 in mice.

[0084] Example 14 Compared with Example 4, the concentration of vitamin D or its derivatives in the single and combined groups was adjusted (the concentrations in the single and combined groups were the same, and the specific dosages are shown in Table 13). The injection concentration of caspofungin in both the single and combined groups was 0.3 mg / kg, and the antibacterial results are shown in Table 13.

[0085] Table 13 Synergistic antifungal effects of caspofungin and vitamin D or its derivatives

[0086] The concentrations of vitamin D or its derivatives were adjusted according to commonly used clinical dosages. Table 13 shows that caspofungin exhibits synergistic antifungal effects at vitamin D2 or D3 concentrations of 0.03–0.3 μg / kg, at calciferol concentrations of 0.003–0.03 μg / kg, at calcidiol concentrations of 0.1–1 μg / kg, at calcidiol monohydrate concentrations of 0.1–1 μg / kg, at alfacalcidol concentrations of 0.001–0.06 μg / kg, at inexciferol concentrations of 0.01–0.1 μg / kg, and at calcipotriol concentrations of 0.01–0.1 μg / kg. All of these formulations exhibited synergistic antifungal effects at concentrations of calcipotriol monohydrate (0.01–0.1 μg / kg), calcitriol (0.001–0.03 μg / kg), masalcalcitriol (0.01–0.1 μg / kg), ircalcitriol (0.01–0.1 μg / kg), paricalcitriol (0.01–0.1 μg / kg), idecalcitriol (0.01–0.1 μg / kg), tacalcitriol (0.03–0.3 μg / kg), and faricalcitriol (0.03–0.3 μg / kg). Both have a synergistic antifungal effect.

[0087] The injection concentration of vitamin D or its derivatives is adjusted according to the commonly used clinical dosage, as shown in Table 13: Caspofungin (fixed dose 0.3 mg / kg) showed significant synergistic antifungal effects with the following vitamin D derivatives within specific concentration ranges (mass ratios refer to [caspofungin: vitamin D derivative]): Vitamin D2 / D3: Concentration 0.03~0.3 μg / kg (mass ratio approximately 10,000:1 ~ 1,000:1); Calciferol: concentration 0.003~0.03 μg / kg (mass ratio approximately 100,000:1 ~ 10,000:1); Calcidiol / calcidiol monohydrate: concentration 0.1~1 μg / kg (mass ratio approximately 3,000:1 ~ 300:1); Alfacalcidol: Concentration 0.001~0.06 μg / kg (mass ratio approximately 300,000:1 ~ 5,000:1); Inecalciferol: concentration 0.01~0.1 μg / kg (mass ratio approximately 30,000:1 ~ 3,000:1); Calcipotriol / Calcipotriol Monohydrate: Concentration 0.01~0.1 μg / kg (mass ratio approximately 30,000:1 ~ 3,000:1); Calcitriol: concentration 0.001~0.03 μg / kg (mass ratio approximately 300,000:1 ~ 10,000:1); Massacalciferol: Concentration 0.01~0.1 μg / kg (mass ratio approximately 30,000:1 ~ 3,000:1); Ircaltriol: Concentration 0.01~0.1 μg / kg (mass ratio approximately 30,000:1 ~ 3,000:1); Paricalcitol: Concentration 0.01~0.1 μg / kg (mass ratio approximately 30,000:1 ~ 3,000:1); Idecalciferol: concentration 0.01~0.1 μg / kg (mass ratio approximately 30,000:1 ~ 3,000:1); Tacalcitol: Concentration 0.03~0.3 μg / kg (mass ratio approximately 10,000:1 ~ 1,000:1); Faricalcitol: Concentration 0.03~0.3 μg / kg (mass ratio approximately 10,000:1 ~ 1,000:1).

[0088] In summary, this invention demonstrates the versatility of the composition across a broad range of synergistic effects by combining various vitamin D derivatives with different types of (1,3)-β-D-glucan synthase inhibitors.

[0089] Example 15: Validation of the synergistic antifungal mechanism To elucidate the molecular mechanism by which the antimicrobial peptide LL-37 produces a synergistic effect when combined with a (1,3)-β-D-glucan synthase inhibitor, this invention employs a variety of physicochemical characterization methods for systematic verification.

[0090] 15.1 Experimental Materials and Methods Strains and culture conditions: Candida albicans SC5314 was cultured in YPD medium at 30°C until the logarithmic growth phase.

[0091] Test drug: Caspofungin (final concentration 0.01 μg / mL) Antimicrobial peptide LL-37 (final concentration 1 μg / mL) Experimental Groups: Blank control group (untreated) Caspofungin monotherapy LL-37 Single Use Group Caspofungin + LL-37 combination group 15.2 Zeta potential measurement Methods: Log-phase Candida albicans SC5314 cells were treated with caspofungin (0.01 μg / mL) and / or LL-37 (1 μg / mL) at 30°C for 3 hours, respectively. Cells were collected and resuspended in deionized water. Zeta potentials of each group of cells were measured at room temperature using dynamic light scattering.

[0092] Result: As Figure 15 As shown in Figure a, the fungal cells in the blank control group exhibited a significant negative charge (approximately -15 mV) on their surface, due to the abundance of negatively charged phosphomannan on the fungal cell wall surface. Treatment with caspofungin or LL-37 alone did not result in a significant change in zeta potential. However, the zeta potential in the combined treatment group showed a significant reversal, changing from negative to positive (approximately +5 mV).

[0093] Mechanism Explanation: Caspofungin disrupts the β-glucan network structure of the fungal cell wall by inhibiting (1,3)-β-D-glucan synthase, exposing the previously shielded cell membrane. The positively charged cationic antimicrobial peptide LL-37 (pI≈11) is then adsorbed in large quantities onto the exposed phospholipid bilayer of the cell membrane, resulting in a shift in the overall surface charge from negative to positive. Quantitative analysis of the zeta potential reversal confirms an effective electrostatic interaction between LL-37 and the exposed cell membrane behind the damaged cell wall.

[0094] 15.3 Detection of cell membrane potential depolarization Methods: The membrane potential-sensitive fluorescent probe DiSC3(5) was used for detection. Log-phase Candida albicans SC5314 cells were collected, resuspended in PBS buffer containing 100 mM KCl, and 1 μM DiSC3(5) fluorescent probe was added. Cells were then treated with caspofungin (0.01 μg / mL) and / or LL-37 (1 μg / mL), respectively. Fluorescence intensity changes were detected using a multi-mode microplate reader (excitation wavelength 622 nm, emission wavelength 670 nm).

[0095] Result: As Figure 15As shown in b, the DiSC3(5) fluorescence intensity in the blank control group and the caspofungin monotherapy group remained at baseline, indicating intact cell membrane potential. The fluorescence intensity in the LL-37 monotherapy group was slightly increased. However, the fluorescence intensity in the combination treatment group was significantly increased (approximately 3-fold increase relative to the control group), indicating significant cell membrane depolarization.

[0096] Mechanism Explanation: DiSC3(5) is a lipophilic cationic fluorescent probe that accumulates within the cell membrane under normal membrane potential, resulting in fluorescence self-quenching. When the cell membrane depolarizes, the probe molecules are released extracellularly, increasing fluorescence intensity. The significant increase in fluorescence intensity in the combined group confirms that, under conditions where caspofungin pre-disrupts the cell wall, LL-37 can effectively penetrate the cell membrane and disrupt its normal electrochemical gradient, leading to loss of membrane potential—a key precursor event to cell death.

[0097] 15.4 Morphological observation by transmission electron microscopy (TEM) Methods: Candida albicans SC5314 cells treated in each group were fixed with 2.5% glutaraldehyde, then fixed with 1% osmium tetroxide, dehydrated with graded ethanol, embedded in epoxy resin, ultrathinly sectioned, and stained with uranium acetate / lead citrate. The ultrastructural changes of the cells were then observed using transmission electron microscopy.

[0098] Result: As Figure 15 As shown in c: Blank control group: The cells are typically oval in shape, with clear and intact cell walls of uniform thickness, cell membranes tightly attached to the inner side of the cell walls, uniform electron density in the cytoplasm, and visible normal organelle structures. Caspofungin monotherapy group: The cell wall boundaries became blurred, with local breaks and irregular defects, and the cell wall thickness was uneven, but the overall cell morphology was basically maintained, and there were no obvious abnormalities in the cytoplasm; LL-37 monotherapy group: Cell morphology was similar to that of the control group, cell wall structure was intact, and no obvious ultrastructural changes were observed. Combined treatment group: The cell wall almost completely disintegrated and disappeared, the cell membrane was severely shrunken and invaginated, a large amount of cytoplasm leaked out and presented an "empty shell" state, the electron density was significantly reduced, the cell morphology was severely distorted and deformed, and it presented typical cell death morphological characteristics.

[0099] Mechanism Explanation: TEM images visually demonstrate the synergistic effect of the "double whammy": Caspofungin first weakens the structural integrity of the cell wall (manifesting as blurred boundaries) by inhibiting (1,3)-β-D-glucan synthase, creating a channel for LL-37 entry. LL-37 then penetrates the damaged cell wall, reaches the cell membrane surface, and inserts into the phospholipid bilayer through its amphiphilic α-helical structure, forming ion channels or causing membrane disintegration, ultimately leading to cytoplasmic leakage and cell death.

[0100] 15.5 Accumulation of fluorescently labeled LL-37 on fungal cell membranes Method: Dilute the Candida albicans SC5314 suspension to 2×10⁻⁶. 6 CFUs / mL (RPMI 1640). Participants were divided into a control group, a single-drug group (0.02 μg / mL caspofungin or 1 μM AMP-FITC), and a two-drug combination group. After drug addition, the mixture was incubated at 30°C for 1 h or 3 h, centrifuged, and washed with PBS; then incubated with 5 μM PI staining solution for 20 min. After washing again, the mixture was resuspended in 200 μL PBS. Imaging was performed using a confocal microscope (FITC / PI dual-channel), and data were processed and analyzed using ZEN software.

[0101] Result: As Figure 16 As shown in a: In the single-drug treatment groups, low-dose caspofungin showed only limited antifungal effects, while the binding ability of the peptide to the fungal surface was also weak when LL-37 was used alone. The combination treatment significantly enhanced the accumulation of antimicrobial peptides on the fungal cell membrane, confirming that caspofungin can assist or promote the binding of antimicrobial peptides to the cell membrane.

[0102] 15.6 Detection of the eversion of negatively charged phospholipids on the surface of fungal cells Method: Dilute the Candida albicans SC5314 suspension to 2×10⁻⁶. 6 CFUs / mL (RPMI 1640). A blank control and caspofungin gradient concentration treatment groups were set up (0.005, 0.007, 0.01 μg / mL). After drug addition, the cells were incubated at 30℃ for 3 h; subsequently, they were centrifuged at 3000×g for 5 min, and the precipitate was collected and washed with PBS. The resulting bacterial resuspended in 195 μL Annexin V-FITC binding buffer, followed by the addition of 5 μL Annexin V-FITC and 10 μL PI, and incubated at room temperature in the dark for 10–20 min. Imaging was performed using a confocal microscope (FITC / PI dual-channel), and the images were processed and analyzed using ZEN software.

[0103] Result: As Figure 16 As shown in b: Confocal microscopy revealed that after treatment with caspofungin, the outward extrusion of negatively charged phospholipids on the surface of surviving fungal cells increased in a dose-dependent manner, consistent with our hypothesis.

[0104] 15.8-15.7 Summary This embodiment systematically verified the molecular mechanism of the synergistic antifungal action between the antimicrobial peptide LL-37 and the (1,3)-β-D-glucan synthase inhibitor from a physicochemical perspective through a variety of complementary experimental methods: Zeta potential reversal confirms that LL-37 can effectively adsorb onto the exposed cell membrane surface after cell wall disruption; Membrane potential depolarization confirms that LL-37 disrupts the electrochemical integrity of the cell membrane; TEM ultrastructural observations provided a clear view of the cell wall disintegration and cytoplasmic leakage caused by the combined use of these methods. Fluorescently labeled LL-37 confirmed that caspofungin can promote the aggregation of LL-37 on the fungal cell membrane; Caspofungin induces the outward turning of negatively charged phospholipids on the surface of fungal cells, and this outward turning of negatively charged phospholipids exhibits a dose-response relationship with caspofungin. Furthermore, the negatively charged phospholipids promote the focusing of LL-37 on the fungal cell membrane as observed above. Furthermore, in our other experiments (results not provided), we found that this phenomenon of negatively charged phospholipids turning outward to promote the aggregation of positively charged LL-37 can be neutralized by the addition of exogenous phospholipids. This perfectly validates our proposed synergistic mechanism.

[0105] The above results provide sufficient experimental verification for the technical solution proposed in this invention, which states that "the antimicrobial peptide LL-37 expression promoter can upregulate the expression of LL-37 in vivo and produce a synergistic lethal effect when used in combination with (1,3)-β-D-glucan synthase inhibitors." This explains why neither drug showed significant activity when used alone in vitro (LL-37 could not penetrate the intact cell wall, and caspofungin at sub-concentrations was not lethal), but produced an unexpectedly strong synergistic bactericidal effect when used in combination.

[0106] In summary, this invention is based on a newly discovered "dual-hit mechanism": 1. Endogenous defense activation (endogenous enhancement of LL-37 expression): Vitamin D or its derivatives act as promoters of the expression of the antimicrobial peptide LL-37, upregulating the expression of the cAMP gene, thereby inducing the synthesis and secretion of large amounts of the endogenous antimicrobial peptide LL-37.

[0107] 2. Barrier breaking and targeted killing: Fungal cell walls typically act as a physical barrier, preventing cationic antimicrobial peptides (such as LL-37) from directly contacting the cell membrane. Therefore, when the cell wall is intact, simply increasing LL-37 levels often fails to produce sufficient antifungal activity (as shown in the examples, calcitriol alone is ineffective).

[0108] (1,3)-β-D-glucan synthase inhibitors (such as echinocandins and triterpenoids) disrupt the integrity of fungal cell walls by inhibiting the synthesis of key cell wall components.

[0109] 3. Collusive lethality: The damaged cell wall exposed the fungal cell membrane, allowing a large amount of LL-37 produced by the antimicrobial peptide LL-37 expression promoter to directly contact and bind to the negatively charged fungal cell membrane (confirmed by the Zeta potential reversal experiment).

[0110] Subsequently, LL-37 leads to increased membrane permeability, membrane potential depolarization (as confirmed by the DiSC3(5) experiment) and an explosion of intracellular reactive oxygen species (ROS), ultimately resulting in fungal cell apoptosis.

[0111] Based on the aforementioned clearly defined biological mechanism, the scope of protection of this invention should not be limited to the compounds of the specific embodiments, but should cover all combinations that produce synergistic effects through this mechanism: Component 1: Endogenous antimicrobial peptide LL-37 expression promoter. Chemically, its superordinate concept is secosteroids, belonging to the vitamin D receptor (VDR) agonists. Compounds of this structural class can effectively induce or upregulate the host's production of endogenous defensive peptides. Its hypoordinate concepts and typical representatives include, but are not limited to, vitamin D and its various pharmaceutically acceptable derivatives.

[0112] The second component consists of (1,3)-β-D-glucan synthase inhibitors, which disrupt cell wall integrity by inhibiting the synthesis of (1,3)-β-D-glucan in the fungal cell wall, thereby assisting LL-37 in contacting and destroying the fungal cell membrane. This class of compounds includes, but is not limited to, echinocandins (cyclic lipopeptide compounds) and triterpenoids.

[0113] This mechanistic complementarity determines the inevitability of their synergistic effect, rather than being a random drug interaction.

[0114] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of antimicrobial peptide LL-37 or an antimicrobial peptide LL-37 expression promoter in combination with (1,3)-β-D-glucan synthase inhibitor in the preparation of antifungal drugs; The mass ratio of the antimicrobial peptide LL-37 to the (1,3)-β-D-glucan synthase inhibitor is (1~5000):1; The mass ratio of the antimicrobial peptide LL-37 expression promoter to the (1,3)-β-D-glucan synthase inhibitor is 1:(1~5×10⁻⁶). 6 ).

2. The application as described in claim 1, characterized in that, The amino acid sequence of the antimicrobial peptide LL-37 is shown in SEQ ID NO: 1; The expression promoter of the antimicrobial peptide LL-37 is vitamin D or its derivative.

3. The application as described in claim 2, characterized in that, The vitamin D or its derivatives include one or more of the following: vitamin D2, vitamin D3, calciferol, calcidiol, calcidiol monohydrate, alfacalciferol, inazacalciferol, calcipotriol, calcipotriol monohydrate, calciferol, masalcalciferol, ercalciferol, paricalciferol, edicalciferol, tacalciferol, and faricalciferol.

4. The application as described in claim 1, characterized in that, The (1,3)-β-D-glucan synthase inhibitors include one or more of echinocandins and triterpenoids; Preferably, the echinocandins include one or more of caspofungin, micafungin, anidoxane, and lezafungin; Preferably, the triterpenoid compound includes irifenol.

5. The application as described in claim 1, characterized in that, The fungi include one or more of Candida albicans, Candida auris, Candida tropicalis, Candida glabrata, Candida parapsilosis, and Candida krusei.

6. An antifungal drug, characterized in that, It includes antimicrobial peptide LL-37 or an antimicrobial peptide LL-37 expression promoter and (1,3)-β-D-glucan synthase inhibitor.

7. The antifungal drug as described in claim 6, characterized in that, The mass ratio of the antimicrobial peptide LL-37 to the (1,3)-β-D-glucan synthase inhibitor is (1~5000):1; The mass ratio of the antimicrobial peptide LL-37 expression promoter to the (1,3)-β-D-glucan synthase inhibitor is 1:(1~5×10⁻⁶). 6 ).

8. The antifungal drug as described in claim 6, characterized in that, The amino acid sequence of the antimicrobial peptide LL-37 is shown in SEQ ID NO: 1; The expression promoter of the antimicrobial peptide LL-37 is vitamin D or a derivative thereof; said vitamin D or its derivative includes one or more of the following: vitamin D2, vitamin D3, ducalciferol, calcidiol, calcidiol monohydrate, alfacalciferol, inalacalciferol, calcipotriol, calcipotriol monohydrate, calciferol, masalcalciferol, ircalciferol, paricalciferol, edicalciferol, tacalciferol, and falicalciferol; The (1,3)-β-D-glucan synthase inhibitors include one or more of echinocandins and triterpenoids; Preferably, the echinocandins include one or more of caspofungin, micafungin, anidoxane, and lezafungin; Preferably, the triterpenoid compound includes irifenol.

9. The antifungal drug as described in claim 6, characterized in that, The fungi include one or more of Candida albicans, Candida auris, Candida tropicalis, Candida glabrata, Candida parapsilosis, and Candida krusei.

10. A pharmaceutical composition, characterized in that, It includes the antifungal drug as described in any one of claims 6 to 9.

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