Antifungal pharmaceutical composition based on (1, 3)-beta-D-glucan synthase inhibitor

Abstract

The invention relates to the technical field of biological medicine, in particular to an antifungal pharmaceutical composition based on a (1, 3)-beta-D-glucan synthase inhibitor. In-vitro experiment results show that the antibacterial peptide LL-37 and (1, 3)-beta-D-glucan synthase inhibitors (caspofungin, micafungin, anidulafungin and iremifungin) are combined for use to achieve the effect of synergistically inhibiting candida albicans, candida auricula, candida tropicalis, candida glabrata, candida parapsilosis and candida krusei, so that the antibacterial peptide LL-37 can be combined with the (1, 3)-beta-D-glucan synthase inhibitors (caspofungin, micafungin, anidulafungin and iremifungin) to achieve the effect of inhibiting the (1, 3)-beta-D-glucan synthase inhibitors). And 3) preparing the antifungal drug by combining the 1, 2, 4, 5, 6, 7, 8, 8, 9, 10- However, the antibacterial peptide LL-37 is easily degraded by in-vivo protease, so that the expression of the antibacterial peptide LL-37 in an organism is up-regulated by adopting an antibacterial peptide LL-37 expression accelerant, and the antibacterial peptide LL-37 and a (1, 3)-beta-D-glucan synthetase inhibitor are synergistically antibacterial in the organism.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an antifungal pharmaceutical composition based on (1,3)-beta-D-glucan synthase inhibitor. BACKGROUND

[0002] Fungal infection, especially invasive fungal infection, has always been a serious threat to human health. Patients receiving organ transplantation, hematopoietic stem cell transplantation, cancer chemotherapy and intravenous catheterization, or infected with HIV virus, can cause the body's immunity to decrease, leading to a high incidence and high mortality of invasive fungal infection. At present, the standard drugs for treating invasive fungal infection in clinical practice mainly include amphotericin B, azoles and lipopeptides. Among them, amphotericin B acts on the lipid of fungal cell membrane and combines with ergosterol, but its kidney toxicity limits its large-scale use. Azole drugs, such as fluconazole and itraconazole, exert antifungal effect by affecting the biosynthesis of fungal cell membrane ergosterol, and are 14alpha-demethylase inhibitors of lanosterol, the main problem of which is serious hepatorenal toxicity and drug resistance.

[0003] (1,3)-beta-D-glucan synthase inhibitor is a new type of antifungal drug, and the currently marketed drugs mainly include echinocandin drugs (such as caspofungin, micafungin, anidulafungin, etc.) and triterpenoids (albaconazole) and the like. (1,3)-beta-D-glucan synthase is a key enzyme for the synthesis of (1,3)-beta-D-glucan in fungal cell wall, and (1,3)-beta-D-glucan synthase inhibitor can destroy the structure of fungal cell wall by blocking the synthesis of (1,3)-beta-D-glucan. Since human cells do not have cell walls, the toxic and side effects of this kind of inhibitor are very low, and it can also be used to treat fungal infections resistant to azole drugs.

[0004] However, long-term use of (1,3)-beta-D-glucan synthase inhibitor can lead to fungal resistance through target enzyme mutation or overexpression, and at the same time, the high price also limits the application of (1,3)-beta-D-glucan synthase inhibitor. SUMMARY

[0005] In order to overcome the above problems, the present application provides an antifungal pharmaceutical composition based on (1,3)-beta-D-glucan synthase inhibitor.

[0006] To achieve the above technical purposes, the present application adopts the following technical solutions: In a first aspect of the present application, the application of antibacterial peptide LL-37 or antibacterial peptide LL-37 expression promoter combined with (1,3)-beta-D-glucan synthase inhibitor in the preparation of antifungal drugs is provided. The mass ratio of the antibacterial peptide LL-37 to the (1,3)-β-D-glucan synthase inhibitor is (1-5000): 1; The mass ratio of the antibacterial peptide LL-37 expression promoter to the (1,3)-β-D-glucan synthase inhibitor is 1: (1-5x10 6 ).

[0007] In one or more embodiments, the mass ratio of the antibacterial peptide LL-37 to the (1,3)-β-D-glucan synthase inhibitor is (1-4000): 1, preferably (5-3200): 1.

[0008] The mass ratio of the antibacterial peptide LL-37 expression promoter to the (1,3)-β-D-glucan synthase inhibitor is 1: (10-4.6x10 6 ), preferably 1: (100-4x10 6 ), and further preferably 1: (1000-3x10 6 ).

[0009] In one or more embodiments, the antibacterial peptide LL-37 is used in combination with the (1,3)-β-D-glucan synthase inhibitor in the preparation of an antifungal drug.

[0010] In one or more embodiments, the antibacterial peptide LL-37 expression promoter is used in combination with the (1,3)-β-D-glucan synthase inhibitor in the preparation of an antifungal drug.

[0011] In one or more embodiments, the amino acid sequence of the antibacterial peptide LL-37 is as shown in SEQ ID NO: 1 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES).

[0012] In one or more embodiments, the antibacterial peptide LL-37 expression promoter is vitamin D or a derivative thereof; Preferably, the vitamin D or a derivative thereof includes one or more of Vitamin D2, Vitamin D3, Doxercalciferol, Calcifediol, Calcifediol Monohydrate, Alfacalcidol, Ergosterol, Inecalcitol, Calcipotriol, Calcipotriol monohydrate, and Calcitriol, and preferably is Calcitriol.

[0013] In one or more embodiments, the (1,3)-beta-D-glucan synthase inhibitor comprises one or more of echinocandins and triterpenoids; Preferably, the echinocandins comprise one or more of caspofungin, micafungin and anidulafungin; Preferably, the triterpenoids comprise albaconazole.

[0014] In one or more embodiments, the fungus comprises one or more of Candida albicans, C. auris, C. tropicalis, C. glabrata, C. parapsilosis and C. krusei.

[0015] In a second aspect of the present application, there is provided an antifungal drug comprising the antimicrobial peptide LL-37 or an antimicrobial peptide LL-37 expression promoter and a (1,3)-beta-D-glucan synthase inhibitor.

[0016] In one or more embodiments, the antifungal drug comprises the antimicrobial peptide LL-37 and a (1,3)-beta-D-glucan synthase inhibitor; The mass ratio of the antimicrobial peptide LL-37 to the (1,3)-beta-D-glucan synthase inhibitor is (1-5000): 1; In one or more embodiments, the antifungal drug comprises the antimicrobial peptide LL-37 expression promoter and a (1,3)-beta-D-glucan synthase inhibitor; The mass ratio of the antimicrobial peptide LL-37 expression promoter to the (1,3)-beta-D-glucan synthase inhibitor is 1:(1-5x10 6 ), preferably 1:(10-4.6x10 6 ), further preferably 1:(100-4x10 6 ), and more further preferably 1:(1000-3x10 6 ).

[0017] In one or more embodiments, the amino acid sequence of the antimicrobial peptide LL-37 is as shown in SEQ ID NO: 1.

[0018] In one or more embodiments, the antimicrobial peptide LL-37 expression promoter is vitamin D or a derivative thereof. Preferably, the vitamin D or its derivative includes one or more of Vitamin D2, Vitamin D3, Doxercalciferol, Calcifediol, Calcifediol Monohydrate, Alfacalcidol, Ergosterol, Inecalcitol, Calcipotriol, Calcipotriol monohydrate and Calcitriol, preferably Calcitriol.

[0019] In one or more embodiments, the (1,3)-β-D-glucan synthesis inhibitor includes one or more of echinocandins and triterpenoids; Preferably, the echinocandins include one or more of Caspofungin, Micafungin and Anidulafungin. Preferably, the triterpenoids include Itraconazole.

[0020] 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.

[0021] In a third aspect, the present application provides a pharmaceutical composition comprising the antifungal drug of the second aspect.

[0022] In one or more embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable carriers, excipients and diluents, etc.

[0023] The non-drug active ingredients that can be included in the carriers, excipients and diluents, etc. are well known in the art, and those of ordinary skill in the art can determine that they meet clinical standards.

[0024] Preferably, the carriers, excipients and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, etc.

[0025] Preferably, the dosage form of the drug is a suspension, an emulsion, a granule, a spray, an injection, a transdermal absorption agent, a dosage form suitable for transfection, a tablet, a powder, a granule or a capsule.

[0026] The drug of the present application can be administered into the body by known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Alternatively, administration is via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration can be via single or multiple doses. The skilled person understands that the actual dose to be administered in the present application can vary greatly depending on a variety of factors, such as the target cells, the biological type or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

[0027] Preferably, the drug administration subject can be human and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, orangutans, and the like.

[0028] The present application has the following beneficial effects: (1) The in vitro experimental results in the present application show that the combination of the antibacterial peptide LL-37 and the (1,3)-β-D-glucan synthase inhibitor (caspofungin, micafungin, anidulafungin and eritoran) has a synergistic inhibitory effect on Candida albicans, Candida auris, Candida tropicalis, Candida glabrata, Candida parapsilosis and Candida krusei, so the antibacterial peptide LL-37 can be combined with the (1,3)-β-D-glucan synthase inhibitor to prepare an antifungal drug. However, the antibacterial peptide LL-37 is easily degraded by proteases in the body, so the present application uses the antibacterial peptide LL-37 expression promoter to up-regulate the expression of the antibacterial peptide LL-37 in the body, and the antibacterial peptide LL-37 in the body cooperates with the (1,3)-β-D-glucan synthase inhibitor to inhibit bacteria.

[0029] (2) The combination of the antibacterial peptide LL-37 or the antibacterial peptide LL-37 expression promoter and the (1,3)-β-D-glucan synthase inhibitor in the present application can effectively reduce the minimum inhibitory concentration of the (1,3)-β-D-glucan synthase inhibitor, reduce the amount of the (1,3)-β-D-glucan synthase inhibitor, and thus overcome the problem of fungal drug resistance in clinical practice.

[0030] (3) The development of an antifungal treatment scheme based on the (1,3)-β-D-glucan synthase inhibitor not only enriches the existing antifungal drug treatment methods, but also provides a new type of effective and safe strategy for the clinical treatment of complex fungal infections, which has important scientific significance and broad application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0031] The drawings accompanying the specification of the present application serve to provide further understanding of the present application, and the illustrative embodiments of the present application and their descriptions serve to explain the present application, and do not constitute an improper limitation on the present application.

[0032] Figure 1Figure showing the effect of LL37 combined with caspofungin against fungi, wherein a is LL37 combined with caspofungin against C. albicans SC5314, b is LL37 combined with caspofungin against C. auris CBS12774; Figure 2 Figure showing the effect of LL37 combined with caspofungin against fungi, wherein a is LL37 combined with caspofungin against C. tropicalis CT2; b is LL37 combined with caspofungin against C. glabrata CG1; Figure 3 Figure showing the effect of LL37 combined with caspofungin against fungi, wherein a is LL37 combined with caspofungin against C. parapsilosis CP1; b is LL37 combined with caspofungin against C. krusei CK1; Figure 4 Figure showing the effect of LL37 combined with micafungin against C. albicans SC5314; Figure 5 Figure showing the effect of LL37 combined with anidulafungin against fungi, wherein a is LL37 combined with anidulafungin against C. albicans SC5314, b is LL37 combined with anidulafungin against C. auris CBS12774; Figure 6 Figure showing the effect of LL37 combined with anidulafungin against fungi, wherein a is LL37 combined with anidulafungin against C. tropicalis CT2; b is LL37 combined with anidulafungin against C. glabrata CG1; Figure 7 Figure showing the effect of LL37 combined with anidulafungin against fungi, wherein a is LL37 combined with anidulafungin against C. parapsilosis CP1; b is LL37 combined with anidulafungin against C. krusei CK1; Figure 8 Figure showing the effect of LL37 combined with eri enconazole against fungi, wherein a is LL37 combined with eri enconazole against C. albicans SC5314, b is LL37 combined with eri enconazole against C. auris CBS12774; Figure 9 Figure showing the effect of LL37 combined with eri enconazole against fungi, wherein a is LL37 combined with eri enconazole against C. tropicalis CT2; b is LL37 combined with eri enconazole against C. glabrata CG1; Figure 10 Figure showing the effect of LL37 combined with eri enconazole against fungi, wherein a is LL37 combined with eri enconazole against C. parapsilosis CP1; b is LL37 combined with eri enconazole against C. krusei CK1; Figure 11The synergistic anti-fungal infection effect of low-dose Caspofungin and Calcitriol in mice, wherein a is the kidney fungal load of mice treated with low-dose Caspofungin; b is the result of kidney section PAS staining of mice; c is the result of kidney section HE staining of mice. Figure 12 The synergistic anti-fungal infection effect of therapeutic-dose Caspofungin and Calcitriol in mice, wherein a is the kidney fungal load of mice treated with therapeutic-dose Caspofungin; b is the result of kidney section PAS staining of mice; c is the result of kidney section HE staining of mice. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0034] It should be noted that the terms used herein are only intended to describe specific embodiments and are not intended to limit exemplary embodiments according to the present application. As used herein, the singular form is intended to include the plural form unless the context clearly indicates otherwise, and it should also be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of the features, steps, operations, devices, components and / or combinations thereof.

[0035] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with specific examples.

[0036] Antibacterial peptide LL37 was purchased from Anhui Guoping Pharmaceutical Co., Ltd.; Caspofungin (CAP), Micafungin, Anidulafungin and Ibrexafungerp citrate were purchased from Yuan Ye Bio; Vitamin D2, Vitamin D3, Doxercalciferol, Calcifediol, Calcifediol Monohydrate, Alfacalcidol, Ergosterol, Inecalcitol, Calcipotriol, Calcipotriol monohydrate and Calcitriol were all purchased from MCE; Cell RNA extraction kit Beyozol purchased from Bi Yun Tian; SPARKscript ®Ⅱ All-in-one RT SuperMix for qPCR Reverse transcription kit purchased from Synergy; Pfeiffer: human diffuse large B-cell lymphoma.

[0037] Example 1 The overnight cultured fungal cells were diluted to 1×10 3 CFU / mL with RPMI 1640 medium, and the diluted bacterial solution was used to prepare working solutions of different drug concentration gradients of caspofungin, micafungin, anidulafungin, and eri enconazole; in the 96-well plate, different concentrations of working solutions were added to each column, and a certain concentration of LL37 antibacterial peptide working solution was added to each well in rows A-G and gradient dilution was performed; after incubation in a 30℃ incubator for 24 h, the OD value of each well was detected at 600 nm wavelength, and the control well was the blank bacterial solution well without drug addition. The growth rate of each well = drug-added well value / control well value × 100%, and the growth rate of the control well was 100%. The drug concentration of the well with an absorbance decrease of 80% compared with the control well was defined as the minimum inhibitory concentration (MIC).

[0038] Synergistic effect evaluation: The combination of antibacterial drugs may exhibit "irrelevant", "additive", "synergistic", and "antagonistic" effects in vitro or in animals. The results of combined drug sensitivity test are judged by the fractional inhibitory concentration index.

[0039] Fractional inhibitory concentration index method (FICI), formula as follows: ΣFICI=FIC A +FIC B =C A / MIC A +C B / MIC B ; Wherein, FICI is the fractional inhibitory concentration index, FIC A and FIC B respectively represent the MIC value of drug A and B when used in combination, divided by the MIC value of drug A and B when used alone, MIC A and MIC B are the minimum inhibitory concentrations of drugs A and B alone, C A and C B are the respective concentrations when the two drugs are used in combination to achieve the same efficacy. FICI>4 is antagonistic effect, FICI between 0.5 and 4 is additive or irrelevant effect, and FICI≤0.5 is defined as synergistic effect.

[0040] The antifungal effect of the combination of the antimicrobial peptide LL37 with caspofungin, micafungin, anidulafungin and eritoran is shown in Figures 1-10 and Tables 1-2.

[0041] Table 1 Results of the combination

[0042] Table 2 Results of the combination

[0043] As shown in Tables 1 and 2, the antimicrobial peptide LL37 alone had no inhibitory effect against Candida albicans SC5314, Candida auris CBS12774, Candida tropicalis CT2, Candida glabrata CG1, Candida parapsilosis CP1, and Candida krusei CK1, with minimum inhibitory concentrations greater than 128 μg / mL. The minimum inhibitory concentrations 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 the antimicrobial peptide LL37 was used in combination with caspofungin, the FICI was less than 0.5, indicating that caspofungin and the antimicrobial peptide LL37 had a significant synergistic effect. When used in combination with the antimicrobial peptide LL37, the minimum inhibitory concentrations of caspofungin against Candida albicans SC5314, Candida auris CBS12774, Candida tropicalis CT2, Candida glabrata CG1, Candida parapsilosis CP1, and Candida krusei CK1 reached 0.015626 μg / mL. The minimum inhibitory concentration of micafungin against Candida albicans SC5314 was 0.03125 μg / mL when used alone. When the antimicrobial peptide LL37 and micafungin were used in combination, the FICI was less than 0.5, indicating that micafungin and the antimicrobial peptide LL37 had a significant synergistic effect. When used in combination with the antimicrobial peptide LL37, the minimum inhibitory concentration of micafungin against Candida albicans SC5314 could reach 0.001953 μg / mL. The minimum inhibitory concentrations of anidulafungin 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 the antibacterial peptide LL37 and anidulafungin are combined, the FICI is less than 0.5, indicating that anidulafungin and antibacterial peptide LL37 have obvious synergies. In the case of combination with antibacterial peptide LL37, the minimum inhibitory concentration of anidulafungin on Candida albicans SC5314 can reach 0.001953 μg / mL, on Candida auris CBS12774 can reach 0.001953 μg / mL, on Candida tropicalis CT2 can reach 0.001953 μg / mL, on Candida glabrata CG1 can reach 0.001953 μg / mL, on Candida parapsilosis CP1 can reach 0.0625 μg / mL, and on Candida krusei CK1 can reach 0.125 μg / mL. The minimum inhibitory concentration of anidulafungin alone on Candida albicans SC5314, Candida auris CBS12774, Candida tropicalis CT2, Candida glabrata CG1, Candida parapsilosis CP1 and Candida krusei CK1 is 0.125 μg / mL, 1 μg / mL, 1 μg / mL, 2 μg / mL, 1 μg / mL and 2 μg / mL, respectively. When the antibacterial peptide LL37 and anidulafungin are combined, the FICI is less than 0.5, indicating that anidulafungin and antibacterial peptide LL37 have obvious synergies. In the case of combination with antibacterial peptide LL37, the minimum inhibitory concentration of anidulafungin on Candida albicans SC5314 can reach 0.0078125 μg / mL, on Candida auris CBS12774 can reach 0.0625 μg / mL, on Candida tropicalis CT2 can reach 0.125 μg / mL, on Candida glabrata CG1 can reach 0.25 μg / mL, on Candida parapsilosis CP1 can reach 0.125 μg / mL, and on Candida krusei CK1 can reach 0.25 μg / mL. In summary, the combination of antibacterial peptide LL-37 and (1,3)-β-D-glucan synthase inhibitors (capofungin, micafungin, anidulafungin and eriaffenine) can effectively reduce the minimum effective inhibitory concentration of (1,3)-β-D-glucan synthase inhibitors, reduce the amount of (1,3)-β-D-glucan synthase inhibitors, and thus overcome the clinical fungal drug resistance problem.

[0044] Example 2 The antibacterial peptide LL-37 is an active peptide generated by enzymatic cleavage of the precursor protein hCAP-18 (human cationic antibacterial protein 18) encoded by the cyclic adenosine monophosphate (CAMP) gene, and belongs to the only human member in the Cathelicidin family. The antibacterial peptide LL-37 is expressed in various types of cells, especially neutrophils, keratinocytes and epithelial cells, and is mainly stored in the form of granular protein in cells, and the active peptide is released by removing the Cathelin domain by serine protease 3 when activated. However, the antibacterial peptide LL-37 is easily degraded by proteases in the body, so the antibacterial peptide LL-37 expression promoter is used to up-regulate the expression of the antibacterial peptide LL-37 in the body.

[0045] Screening of antibacterial peptide LL-37 expression promoter: The experimental process is as follows: (1) Sample preparation: adjust Pfeiffer cells in logarithmic growth phase to a density of 2 x 10 5 cells / mL; inoculate in a 24-well plate, add 100 nM concentration of test drugs to each well, and set up a blank control group; place the 24-well plate in a 37 °C incubator containing 5% CO2 for 24 h; (2) RNA extraction: after incubation, extract cell RNA according to the method of the Beyozol kit; (3) Reverse transcription: reverse transcribe RNA into cDNA using SPARKscript ®Ⅱ All-in-one RT SuperMix for qPCR reverse transcription kit; (4) Real-time fluorescent quantitative PCR: use β-actin as an internal standard gene, and use the Eppendorf Mastercycler real-time quantitative PCR system to determine the expression of the CAMP gene; 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. It can be seen from Table 3 that vitamin D or its derivatives can increase the expression level of the CAMP gene in the Pfeiffer cell line to varying degrees, among which the CAMP gene expression level in the Pfeiffer cell line induced by calcitriol is the highest.

[0046] Table 3 Effects of vitamin D or its derivatives on CAMP gene expression

[0047] Example 3 Low-dose caspofungin and calcitriol synergistically protect against fungal infections in mice: Eight-week-old Balb / c male mice were randomly divided into four groups, with three mice in each group: low-dose caspofungin alone group, calcitriol alone group, combination group, and control group. Overnight cultured Candida albicans SC5314 cells were diluted to 5×10 6 Each mouse was injected with 100 μL of cell suspension via the tail vein. 24 hours after infection, the different drugs were injected intraperitoneally. Based on body weight, mice were injected with 0.1 mg / kg of caspofungin in the caspofungin alone group, 1 μg / kg of calcitriol in the calcitriol alone group, and a mixed solution of 0.1 mg / kg caspofungin and 1 μg / kg calcitriol in the combination group. The control group received an equal volume of saline. Dosing was continued once daily for three consecutive days. On the fourth day, mice were euthanized, and their kidneys were dissected. One kidney was fixed in 4% paraformaldehyde and sectioned for post-operative scintigraphy and hematoxylin and eosin staining. The other kidney was ground and plated to calculate the bacterial load.

[0048] The experimental results are as follows Figure 11 As shown, from Figure 11 It can be seen that 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, when used in combination with calcitriol, the bacterial load in the kidney is significantly reduced compared with the low-dose caspofungin monotherapy group, the control group, and the calcitriol monotherapy group, indicating that when caspofungin does not produce a therapeutic effect in vivo, its combination with calcitriol can produce a therapeutic effect.

[0049] Example 4 Therapeutic doses of caspofungin and calcitriol synergistically protect against fungal infections in mice: Eight-week-old Balb / c male mice were randomly divided into four groups, with three mice in each group: caspofungin alone group, calcitriol alone group, combination group, and control group. Overnight cultured Candida albicans SC5314 cells were diluted to 5×10 6CFU / mL; 100 μL of cell suspension was injected into each mouse via the tail vein. 24 hours after infection, different drugs were injected intraperitoneally. Mice received doses based on body weight: 0.3 mg / kg caspofungin in the caspofungin alone group, 1 μg / kg calcitriol in the calcitriol alone group, and a mixed solution of 0.3 mg / kg caspofungin and 1 μg / kg calcitriol in the combination group. The control group received an equal volume of saline. Dosing was repeated once daily for three consecutive days. On the fourth day, mice were euthanized, and their kidneys were dissected. One kidney was fixed in 4% paraformaldehyde and sectioned for post-operative smear analysis and hematoxylin and eosin staining; the other kidney was ground and plated to calculate the bacterial load.

[0050] The experimental results are as follows Figure 12 As shown, from Figure 12 It can be seen that calcitriol can significantly enhance the antifungal activity of caspofungin against Candida albicans SC5314 in vivo. The therapeutic dose of caspofungin is the concentration of caspofungin commonly used in laboratory treatment. After combined use with calcitriol, the renal bacterial load was significantly reduced compared with the caspofungin monotherapy group, calcitriol monotherapy group and blank group, indicating that while caspofungin produces a normal therapeutic effect, the effect is enhanced after combined use with calcitriol, which can achieve a synergistic effect.

[0051] Example 5 Therapeutic doses of caspofungin and calcitriol synergistically protect against fungal infections in mice: Compared with Example 4, the fungi in the mice were replaced with Candida auris CBS12774, Candida tropicalis CT2, Candida glabrata CG1, Candida parapsilosis CP1, and Candida krusei CK1. The dosage of caspofungin alone, calcitriol, combination, and control groups was the same as that in Example 4. The other experimental procedures were exactly the same as those in Example 4. The antibacterial results are shown in Table 4.

[0052] Table 4 Synergistic antifungal effect of caspofungin and calcitriol

[0053] Example 6 Therapeutic doses of caspofungin and vitamin D or its derivatives synergistically protect against infection in mice: Compared with Example 4, the fungus in the mouse is still C. albicans SC5314, calcipotriol is replaced by other vitamin D or its derivatives, the injection concentration of caspofungin in the caspofungin single use group is 0.3 mg / kg, the injection concentration of vitamin D or its derivatives in the vitamin D or its derivatives single use group is 1 μg / kg, the injection concentration of the mixture of 0.3 mg / kg caspofungin and 1 μg / kg vitamin D or its derivatives in the combination group, and the injection concentration of the same volume of normal saline in the control group; other experimental processes are exactly the same as those in Example 4; the antibacterial results are shown in Table 5.

[0054] Table 5 Synergistic antifungal effect of caspofungin and vitamin D or its derivatives

[0055] Example 7 Synergistic antifungal effect of therapeutic dose micafungin and vitamin D or its derivatives against infection in mice: Compared with Example 4, the fungus in the mouse is still C. albicans SC5314, caspofungin is replaced by micafungin, the injection concentration of micafungin in the micafungin single use group is 0.3 mg / kg, the injection concentration of vitamin D or its derivatives in the vitamin D or its derivatives single use group is 1 μg / kg, the injection concentration of the mixture of 0.3 mg / kg micafungin and 1 μg / kg vitamin D or its derivatives in the combination group, and the injection concentration of the same volume of normal saline in the control group; other experimental processes are exactly the same as those in Example 4; the antibacterial results are shown in Table 6.

[0056] Table 6 Synergistic antifungal effect of micafungin and vitamin D or its derivatives

[0057] Example 8 Synergistic antifungal effect of therapeutic dose anidulafungin and vitamin D or its derivatives against infection in mice: Compared with Example 4, the fungus in the mouse is still C. albicans SC5314, caspofungin is replaced by anidulafungin, the injection concentration of anidulafungin in the anidulafungin single use group is 0.3 mg / kg, the injection concentration of vitamin D or its derivatives in the vitamin D or its derivatives single use group is 1 μg / kg, the injection concentration of the mixture of 0.3 mg / kg anidulafungin and 1 μg / kg vitamin D or its derivatives in the combination group, and the injection concentration of the same volume of normal saline in the control group; other experimental processes are exactly the same as those in Example 4; the antibacterial results are shown in Table 7.

[0058] Table 7 Synergistic antifungal effect of anidulafungin and vitamin D or its derivatives

[0059] Example 9 Therapeutic dose anidulafungin and calcitriol synergistically against fungal infection in mice in vivo: Compared with Example 8, the fungus in mice in vivo was replaced by C. auris CBS12774, C. tropicalis CT2, C. glabrata CG1, C. parapsilosis CP1 and C. krusei CK1, the injection concentration of anidulafungin in the anidulafungin single use group was 0.3 mg / kg, the injection concentration of calcitriol in the calcitriol single use group was 1 μg / kg, the injection concentration of the mixed solution of 0.3 mg / kg anidulafungin and 1 μg / kg calcitriol in the combination group, and the same volume of normal saline was injected in the control group; the other experimental processes were exactly the same as those in Example 4; the antibacterial results are shown in Table 8.

[0060] Table 8 Anidulafungin and calcitriol synergistically against fungi

[0061] Example 10 Therapeutic dose anidulafungin and calcitriol synergistically against fungal infection in mice in vivo: Compared with Example 4, the fungus in mice in vivo was still C. albicans SC5314, and caspofungin was replaced by anidulafungin, the injection concentration of anidulafungin in the anidulafungin single use group was 0.3 mg / kg, the injection concentration of vitamin D or its derivative in the vitamin D or its derivative single use group was 1 μg / kg, the injection concentration of the mixed solution of 0.3 mg / kg anidulafungin and 1 μg / kg vitamin D or its derivative in the combination group, and the same volume of normal saline was injected in the control group; the other experimental processes were exactly the same as those in Example 4; the antibacterial results are shown in Table 9.

[0062] Table 9 Anidulafungin and calcitriol synergistically against fungi

[0063] Example 11 Therapeutic dose anidulafungin and calcitriol synergistically against fungal infection in mice in vivo: Compared with Example 10, the fungus in mice in vivo was replaced by C. auris CBS12774, C. tropicalis CT2, C. glabrata CG1, C. parapsilosis CP1 and C. krusei CK1, the injection concentration of anidulafungin in the anidulafungin single use group was 0.3 mg / kg, the injection concentration of calcitriol in the calcitriol single use group was 1 μg / kg, the injection concentration of the mixed solution of 0.3 mg / kg anidulafungin and 1 μg / kg calcitriol in the combination group, and the same volume of normal saline was injected in the control group; the other experimental processes were exactly the same as those in Example 4; the antibacterial results are shown in Table 10.

[0064] Table 10 Anidulafungin and calcitriol synergistically against fungi

[0065] As can be seen from the results in Examples 4 to 11, (1,3)-β-D-glucan synthase inhibitors, including caspofungin, micafungin, anidulafungin and eritoran, can be used in combination with vitamin D or its derivatives, including vitamin D2, vitamin D3, doxercalciferol, calcifediol, calcifediol monohydrate, alfacalcidol, calcipotriol and calcitriol, to achieve a synergistic antifungal effect, wherein calcitriol is the most preferred in the same dosage treatment. Caspofungin, micafungin, anidulafungin and eritoran can all be used in combination with calcitriol to reduce the fungal load of C. albicans SC5314, C. auris CBS12774, C. tropicalis CT2, C. glabrata CG1, C. parapsilosis CP1 and C. krusei CK1 in mice.

[0066] Example 12 Compared with Example 4, the concentration of vitamin D or its derivatives in the single use group and the combination use group was adjusted (the concentration in the single use group and the combination use group was the same, and the specific dosage is shown in Table 11), and the injection concentration of caspofungin in the single use group and the combination use group was 0.3 mg / kg, and the antibacterial results are shown in Table 11.

[0067] Table 11 Caspofungin and vitamin D or its derivatives synergistic antifungal

[0068] According to the commonly used dose in clinic, the injection concentration of vitamin D or its derivatives was adjusted, and it can be seen from Table 11 that caspofungin can have a synergistic antifungal effect when the concentration of vitamin D2 or D3 is between 0.03 and 0.3 μg / kg, when the concentration of doxercalciferol is between 0.003 and 0.03 μg / kg, when the concentration of calcifediol is between 0.1 and 1 μg / kg, when the concentration of calcifediol monohydrate is between 0.1 and 1 μg / kg, when the concentration of alfacalcidol is between 0.001 and 0.06 μg / kg, and when the concentration of calcitriol is between 0.001 and 0.03 μg / kg.

[0069] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of antimicrobial peptide LL-37 or an antimicrobial peptide LL-37 expression promoter combined 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 ).

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

3. The use according to claim 2, characterized in that The vitamin D or its derivatives include: one or more of vitamin D2, vitamin D3, doxorcalciferol, calcifediol, calcifediol monohydrate, alfacalcidol, ergosterol, inacalcitol, calcipotriol, calcipotriol monohydrate and calcitriol.

4. The use according to claim 1, wherein The (1,3)-β-D-glucan synthase inhibitor includes one or more of the echinocandins and triterpenoids; The echinocandin drugs include one or more of caspofungin, micafungin and anidulafungin; The triterpenoid compounds include erefungin.

5. The use according to 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: The invention comprises antimicrobial peptide LL-37 or an antimicrobial peptide LL-37 expression promoter and a (1,3)-β-D-glucan synthase inhibitor.

7. The antifungal drug according to claim 6, wherein 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 ).

8. The antifungal drug according to claim 6, wherein The amino acid sequence of the antimicrobial peptide LL-37 is shown in SEQ ID NO: 1; The antimicrobial peptide LL-37 expression promoter is vitamin D or its derivatives; the vitamin D or its derivatives include: one or more of vitamin D2, vitamin D3, doxorcalciferol, calcifediol, calcifediol monohydrate, alfacalcidol, ergosterol, inecalcitol, calcipotriol, calcipotriol monohydrate and calcitriol; The (1,3)-β-D-glucan synthase inhibitor includes one or more of the echinocandins and triterpenoids; The echinocandin drugs include one or more of caspofungin, micafungin and anidulafungin; The triterpenoid compounds include erefungin.

9. The antifungal drug according to claim 6, wherein 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 comprises the antifungal drug according to any one of claims 6 to 9.

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